TGF-alpha polypeptides, functional fragments and methods of use therefor

ABSTRACT

Disclosed are peptides related to human TGF-α, having TGF-α biological activity, which are useful for many of the indications that full-length TGF-α polypeptide is useful. Also provided are methods of use of such peptides, as well as human TGF-α and biologically related polypeptides. For example, methods for treating or preventing cachexia in subjects are provided as well as methods for stimulating hematopoiesis in patients undergoing cytotoxic chemotherapy. In addition, the use of TGF-α related peptides to related neurodengenerative diseases is also provided. Methods of the invention also provide protection for patients undergoing cytotoxic therapy from side effects such as gastrointestinal (GI) mucositis.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 09/459,813, filed Dec. 13, 1999, which is acontinuation-in-part of U.S. application Ser. No. 09/299,473, filed Apr.26, 1999, the disclosures of which are herein incorporated by referencein their entirety.

FIELD OF THE INVENTION

[0002] The invention relates generally to transforming growth factoralpha ( ) and more specifically to methods of using TGF-α forstimulating hematopoiesis, for suppressing immune function associatedwith autoimmune diseases, for suppressing inflammatory responsesmediated by excessive histamine release and by expression ofTNF-receptors and associated pro-inflammatory cytokines, and fortreating cachexia or for treating or preventing mucositis andgastrointestinal-associated disorders.

BACKGROUND

[0003] There are several disease treatments that could significantlybenefit by having cells regenerate after injury or lesion formation,particularly in the CNS, in the immune system and in thegastrointestinal tract. In some instances, a particular treatment for adisease often detrimentally affects the subject being treated. Forexample, administration of chemotherapeutic agents to subjects resultsin destruction of healthy cells, for example, cells of thegastrointestinal tract. A number treatment-related disorders are relatedto the choice of chemotherapeutic agent. Such agents include carmustine(BCNU), chlorambucil (Leukeran), cisplatin (Platinol), Cytarabine,doxorubicin (Adriamycin), fluorouracil (5-FU), methoxetrate (Mexate),taxol, CPT111, etoposide, and plicamycin (Mithracin) which are known fortheir direct stomatotoxic potential (Sonis, 1993, “Oral Complications inCancer Therapy,” In: Principles and Practice of Oncology, pp. 2385-2394,DeVitta et al., Eds., J. B. Lippincott, Philadelphia) and henceincidence of mucositis.

[0004] Oral mucositis can be initiated by the cytotoxic effects ofchemotherapy and/or radiotherapy on the rapidly dividing epithelialcells of the oropharyngeal mucosa, and is exacerbated by infection withboth endogenous oral flora and opportunistic bacterial and fungalpathogens. Complications related to oral mucositis vary in the differentpatient populations affected, but typically include pain, poor oralintake with consequent dehydration and weight loss, and systemicinfection with organisms originating in the oral cavity. The painassociated with oral mucositis may be severe requiring narcoticanalgesics, and the difficulty in eating can result in patientsreceiving total parenteral nutrition.

[0005] Current therapies have been directed at decreasing oral flora andthe extent of infection of oral ulcerations. Systemic treatment with G-and GM-CSF has been shown to result in a decreased incidence of oralmucositis, presumably by allowing for more rapid neutrophil recovery andthus an improved ability to combat infection, although it has beenpostulated that the CSFs may have a more direct effect on the oralmucosa (Chi et al., 1995, J. Clin. Oncol. 13:2620-2628). In one study,GM-CSF was reported to exacerbate mucositis. (Cartee et al., 1994,Cytokine 7:471-477). Benzydamine hydrochloride, a nonsteroidal drug withanalgesic and antimicrobial properties, has been studied both inpatients undergoing radiation therapy and in patients receivingintra-arterial chemotherapy.

[0006] In addition, diseases associated with epithelial cell depletionin the gastrointestinal tract often increase the risk of relateddisorders. Such related disorders include infection by opportunisticpathogens as well as weight loss associated with the loss in nutrientuptake in the gastrointestinal tract.

SUMMARY OF THE INVENTION

[0007] The present invention is based on the seminal discovery thatTGF-α and functionally related polypeptides, TGF-α mimetics, functionalTGF-α peptides, and polynucleotides encoding such polypeptides andpeptide fragments are effective for treating or preventing weight-lossin subjects having disorders or diseases associated with weight-loss(e.g., cachexia). In addition, the polypeptides of the invention havemitogenic and barrier function (e.g., protective) effects on stem cellsand their differentiated progeny from a variety of tissues including thegastrointestinal system, the nervous system and the hematopoieticsystem.

[0008] In a first embodiment, the invention provides a method oftreating a subject having or at risk of having cachexia comprisingadministering to the subject a transforming growth factor-alpha (TGF-α)polypeptide in an amount effective to prevent or reduce weight-loss. Inone aspect, the invention provides a method of increasing the bodyweight of a subject comprising administering to the subject, prior to,simultaneously with, or substantially following chemotherapy, atransforming growth factor-alpha (TGF-α) polypeptide in an amounteffective to increase the weight of the subject. In one aspect thesubject has AIDS related complex (ARC) or AIDS and cachexia associatedwith such diseases.

[0009] In another embodiment, the invention provides a method fortreating or preventing mucositis of the gastrointestinal tract in asubject, comprising administering a TGFα or related polypeptide in anamount effective to treat or prevent mucositis in the subject. Forexample, a subject undergoing chemotherapy can be treated by the methodof the invention.

[0010] The invention also provides a polypeptide comprising a peptidehaving a sequenceNH₂-X_(1a)-Cys-His-Ser-X_(1b)-X₂-X_(1a)-X_(1b)-X_(1a)-X₃-Cys-COOH (SEQID NO:4) wherein X_(1a) and X_(1b) are independently Val, Gly or Ala; X₂is Tyr or Phe; X₃ is Arg or Lys; and the two Cys moieties are linked viaa disulfide bond to form an at least 11-amino acid functional peptidehaving TGF-α activity.

[0011] In another embodiment, the invention provides a polypeptidecomprising a peptide having a sequenceNH₂-X_(1a)-Cys-His-Ser-X_(1b)-X₂-X_(1a)-X_(1b)-X_(1a)-X₃-Cys-COOH (SEQID NO:4) wherein X_(1a) and X_(1b) are independently Val, Gly or Ala; X₂is Tyr or Phe; X₃ is Arg or Lys; and the two Cys moieties are linked viaa disulfide bond to form an at least 11-amino acid functional peptidehaving TGF-α activity and wherein at least one or more of the followingamino acids are linked to the C-terminal Cys moiety of SEQ IDNO:4:-X₄-His-X_(1c)-X₄-X₅- X₆-X_(1c) (SEQ ID NO:5) wherein X₄ is Glu orAsp; X₅ is Leu or Ile; and X₆ is Asp or Glu.

[0012] In yet another embodiment, the invention provides apharmaceutical composition comprising a polypeptide having a sequenceNH₂-X_(1a)-Cys-His-Ser-X_(1b)-X₂-X_(1a)-X_(1b)-X_(1a)-X₃ -Cys COOH (SEQID NO:4) wherein X_(1a) and X_(1b) are independently Val, Gly or Ala; X₂is Tyr or Phe; X₃ is Arg or Lys; and the two Cys moieties are linked viaa disulfide bond to form an at least 11-amino acid functional peptidehaving TGF-α activity, and a pharmaceutically acceptable carrier. Inaddition, at least one or more of the following amino acids are linkedto the C-terminal Cys moiety of SEQ IDNO:4:-X₄-His-X_(1c)-X₄-X₅-X₆-X_(1c) (SEQ ID NO:5) wherein X4 is Glu orAsp; X₅ is Leu or Ile; and X₆ is Asp or Glu.

[0013] The invention also provides a compound that acts as a TGF-αmimetic, comprising a compound of formula: loop peptideN-terminus-linker-cyclic C₄H₈N₂-linker-loop peptide N-terminus whereinthe linker moiety is designed to link the N-terminus of the loop peptideto a nitrogen atom of the ring C₄H₈N₂ and wherein the loop peptide has asequence NH₂-X_(1a)-Cys-His-Ser-X_(1b)-X₂-X_(1a)-X_(1b)-X_(1a)-X₃-CysCooh (SEQ ID NO:2) wherein X_(1a) and X_(1b) are independently Val, Glyor Ala; X₂ is Tyr or Phe; X₃ is Arg or Lys; and the two Cys moieties arelinked via a disulfide bond to form an at least 11-amino acid functionalpeptide having TGF-α activity.

BRIEF DESCRIPTION OF THE FIGURE

[0014]FIG. 1 shows the structure of rat TGFα polypeptide and its 50amino acids arranged into three loops (SEQ ID NO:2). The human TGFαsequence is provided in SEQ ID NO:1 with a similar tertiary structureand a close sequence homology.

[0015]FIG. 2 shows a graph comparing TGFα biological activity of thethree loop peptide regions of TGFα (see FIG. 1) wherein Loop A is aminoacids 1-21 (starting at the N terminus), Loop B is amino acids 16 to 32and Loop C is amino acids 33 to 50. Only Loop C showed significant TGFαactivity as determined by cell proliferation and in a dose responsefashion.

[0016]FIG. 3 shows a graph of mouse spleen weights that were treatedwith Cis Platinum (CP) at either 5 μg/g or 10 μg/g and with TGFα atconcentrations of 10 ng/lg or 50 ng/g. These data show that TGFαtreatment caused a return to normal spleen weights despite CP treatmentthat reduced spleen weights significantly.

[0017]FIG. 4 depicts a summary of histological data that measuredaverage crypt height of the three groups of mice. TGFα 57 treatment (50ng/g) was able to more-than-restore crypt height loss from CP treatment.

[0018]FIG. 5 shows a graph depicting the effects of cisplatinum-alone,cisplatinum and a TGF-α polypeptide, and a TGF-α alone on weight loss ofmice.

[0019]FIG. 6 shows weight loss in mice following cisplatinadministration with and without concurrent TGF-α treatment. The graphshows (from left to right) the proportion of weight-loss in the presenceof PBS alone, TGF-α alone, cisplatin alone, and cisplatin+TGF-α.

DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention provides compositions of TGF-αpolypeptides, TGF-α mimetics, TGF-α related polypeptides and functionalfragments thereof as well as polynucleotides encoding the polypeptidesand fragments thereof. In addition, the invention provides methods ofusing the polypeptides and polynucleotides of the invention for treatingor preventing a number of diseases and disorders.

Transforming Growth Factor-α

[0021] TGF-α is a member of the epidermal growth factor (EGF) family andinteracts with one or more receptors in the EGF-family of receptors.TGF-α stimulates the receptor's endogenous tyrosine kinase activitywhich results in activating various cellular functions, such asstimulating a mitogenic or migration response in a wide variety of celltypes. TGF-α and EGF mRNAs reach their highest levels and relativeabundance (compared to total RNA) in the early postnatal period anddecrease thereafter, suggesting a role in embryonic development. From ahistological perspective, TGF-α is found in numerous cell types andtissues throughout the body. The active form of TGF-α is derived from alarger 30-35 kD precursor and contains 50 amino acids. Human TGF-αshares only a 30% structural homology with the 53-amino acid form ofEGF, but includes conservation and spacing of all six cysteine residues.TGFα is highly conserved among species. For example, the rat and humanpolypeptides share about 90% homology compared to a 70% homology asbetween the rat and human EGF polypeptide. The amino acid sequence ofhuman TGFα is shown in SEQ ID NO:1. TGFα shares cysteine disulfide bondstructures with a family of proteins including vaccinia growth factor,amphiregulin precursor, betacellulin precursor, heparin binding EGF-likegrowth factor, epiregulin (rodent only), HUS 19878, myxomavirus growthfactor (MGF), Shope fibroma virus growth factor (SFGF), and schwannomaderived growth factor. Such TGF-α related polypeptides are also usefulin the methods of the invention.

[0022] TGF-α is an acid and heat stable polypeptide of about 5.6 kDamolecular weight. It is synthesized as a larger 30-35 kDa molecularweight glycosylated and membrane-bound precursor protein wherein thesoluble 5.6 kDa active form is released following specific cleavage byan elastase-like protease. TGF-α binds with high affinity in thenanomolar range and induces autophosphorylation of one or more membersof EGF receptor family (e.g., ErbB1 through 4) to transduce subsequentsignal pathways with the EGF receptors. TGF-α is 50 amino acids inlength and has three disulfide bonds to form its tertiary configuration.All three disulfide bonds are required for activity. TGF-α is stored inprecursor form in alpha granules of some secretory cells. Moreover, theprimary amino acid sequence is highly conserved among various speciesexamined, such as more than 92% homology at the amino acid level asbetween human and rat TGFα polypeptides.

[0023] Human TGFα is a polypeptide of 50 amino acids. The correspondingrat sequence is shown in FIG. 1. The human or rat TGFα polypeptide canbe divided roughly into three loop regions corresponding roughly(starting at the N terminus) to amino acids 1-21, to amino acids 16-32,and to amino acids 33-50. As discussed more fully below, the inventionprovides functional fragments of TGF-α that retain TGF-α biologicalactivity. “Functional fragment” as used herein means a TGF-α peptidethat is a fragment or a modified fragment of a full length TGF-αpolypeptide or related polypeptide so long as the fragment retains someTGF-α related biological activity (e.g., interacts with an EGF familyreceptor, stimulates proliferation or migration of stem cells, usefulfor treating or preventing cachexia). Other biological activitiesassociated with the polypeptides of the invention include, for example,mitogenic effects on stem cells and their more differentiated progeny ofvarious tissues (e.g., epithelial stem cells, hematopoietic stem cells,neural stem cells, liver stem cells, keratinocyte stem cells, andpancreatic derived stem cells).

[0024] The mucosal epithelium of the intestine is in a continuallydynamic state known as “epithelial renewal” in which undifferentiatedstem cells from a proliferative crypt zone divide, differentiate andmigrate to the luminal surface. Once terminally differentiated, mucosalepithelial cells are sloughed from the tips of the villi. The turnoverof the crypt-villus cell population is rapid and occurs every 24-72hours. Continuous exfoliation of the cells at the villus tip iscounterbalanced by ongoing proliferation in the crypt so that netintestinal epithelial mass remains relatively constant. Therapidly-proliferating epithelium of the gastrointestinal tract isextremely sensitive to cytotoxic drugs that are widely used in cancerchemotherapy. By “gastrointestinal tract” is meant, for example, thetissues of the mouth, esophagus, stomach, small intestine, largeintestine, rectum and anus. This “side effect” reduces the tolerateddose of such drugs as it can cause a breakdown of the GI barrierfunction and septic propagate a septic condition in a patient alreadyimmuno-compromised. This can also lead to life-threatening hemorrhage.Therefore, there is a need in the art for the development of productsand delivery systems that stimulate the repair and rejuvenation ofmucosal epithelium in the gastrointestinal tract to provide benefit tosubjects having, for example, weight-loss disorders associated withchemotherapy and radiation therapy for cancer as well as disorders ordiseases associated with pathogens such as HIV.

[0025] Accordingly, the invention provides a class of peptides,including TGF-α and those smaller than the 50 amino acid human TGF-α,yet retaining TGF-α biological activity, which are useful aspharmacologic and therapeutic agents. Other polypeptides or fragmentsthereof include TGF-related polypeptides that have the biologicalactivity of TGF-α (e.g., amphiregulin, vaccinia growth factor,myxomavirus growth factor (MGF), Shope fibroma virus growth factor(SFGF), heparin-binding EGF-like growth factor (HB-EGF)).

[0026] The invention also provides methods of using TGF-α, relatedpolypeptides and peptide fragments thereof as disclosed herein tostimulate hematopoiesis in subjects undergoing cytotoxic cancerchemotherapy and to act as a cytoprotective agents and in treatments forsubjects at risk of or having weight-loss disorders associated withcancer cytotoxic therapy. Such disorders include for example,gastrointestinal (GI) mucositis, which can be the result of cytotoxictherapy. While not wanting to be bound to a particular theory, it isbelieved TGF-α may alleviate GI mucositis, in part, through itsmitogenic actvity for GI epithelial stem cells.

[0027] TGFα has been investigated extensively and has recently beenidentified as useful for treating subjects with neurological deficits.This mechanism is thought to stimulate proliferation and migration ofstem cells of neural origin to those sites or lesions in a deficit. Forexample, Parkinson's Disease is characterized by resting tremor,rigidity, inability to initiate movement (akinesia) and slowness ofmovement (bradykinesia). The motor deficits are associated withprogressive degeneration of the dopaminergic innervation to the nucleusaccumbens and degeneration of noradrenergic cells of the locus ceruleusand serotonergic neurons of the raphe. Up to 80% of nigral dopamineneurons can be lost before significant motor deficits are manifest. TGFαwas shown, when infused into rat brains, to be useful for the treatmentof neurodegenerative disorders. Intracerebroventricular (ICV) orintrastriatal infusions of TGFα induced neuronal stem cellproliferation, but degenerating or damaged or otherwise abnormal cellsneeded to be present to facilitate migration of the neuronal stem cellsto a site of injury on a scale sufficient to impact recovery from anassociated neurological deficit. Forebrain neural stem cells, that giverise to migrating progenitor cells that affect treatment and recoveryfrom a neurological deficit disorder, are the migrating cells thataffect treatment recovery from a neural deficit disorder (e.g.,Parkinson's Disease, Huntington's Disease, Alzheimer's Disease and thelike).

[0028] Neural stem cells have been found in subependyma throughout theadult rodent CNS (Ray et al. Soc. Neurosci. 22:394.5, 1996) and in thesubependyma of adult human forebrain (Kirschenbaum et al., CerebralCortex 4:576-589, 1994). Thus, the discovery that TGFα stimulatesproliferation of neural stem cells and promotes migration to a site ofinjury or deficit has led to its investigation for the treatment of aneurodegenerative disorder (Alzheimer's Disease, Huntington's Diseaseand Parkinson's Disease) or CNS traumatic injury (e.g., spinal chordinjury), demyelinating disease, CNS inflammatory disease, CNS autoimmunedisease (e.g., multiple sclerosis) and CNS ischemic disease (e.g.,stroke or brain attack).

[0029] A CNS stem cell has the potential to differentiate into neuronsand astrocytes as well as self replication and thus self renewal. Bothneuronal and glial cells are derived from a common precursor cell. Inthe vertebrate CNS, pluripotential cells have been identified in vitroand in vivo. Certain mitogens, such as TGF-α, can cause proliferation ofCNS pluripotential cells in vitro. Thus, it is possible to harvest suchcell from a subject, treat them ex vivo to stimulate proliferation inculture and then readminister the cells back to a subject.Immunohistochemical analysis in the human brain supports the notion thatTGF-α and its 35 kD precursor are widely distributed in neurons andglial cells both during development and during adulthood. In TGF-αknockout mice genetically altered to lack expression of functioningTGF-α, there was a decrease in neural progenitor cell proliferation inforebrain subependyma, providing evidence for TGF-α as a proliferativefactor for neural progenitor cells.

[0030] TGF-α is found mainly in various neurons of the CNS duringdevelopment and in the adult brain in the cerebral neocortex,hippocampus and striatum. It is also found in glial cells, primarily inthe cerebral and cerebellar cortex areas. Northern blot analyses showedthat TGFα but not EGF (epidermal growth factor) is the most abundantligand that binds to one or more of the EGF receptor family in thebrain. TGFα MRNA levels were 15-170 times higher than EGF in cerebellumand cerebral cortex. TGFα also appears in germinal centers of the brainduring neurogenesis and gliogenesis in the developing brain. In themidbrain, the distribution of TGFα overlaps with tyrosine hydroxylasemRNA and fetal dopaminergic neurons. In culture, TGFα enhanced survivaland neurite outgrowth of neonatal rat dorsal ganglion neurons (EGF didnot) and survival and differentiation of CNS neurons. TGFα inducedproliferation of neural precursor cells of the murine embryonicmesencephalon and further induced a significant increase in the numberof astroglia and microglia in fetal rat medial septal cells. TGFαincreased glutamic acid decarboxylase activity and decreased cholineactetyltransferase activity. Thus, TGFα acted as a general neuronalsurvival factor affecting both cholinergic and GABAergic neurons. Inaddition, TGFα is a mitogen for pluripotent brain stem cells. Forebrainsubependyma contains nestin positive neural stem cells and theirprogeny, which are constitutively proliferating progenitor epithelialcells. A “knockout” mouse that was genetically engineered to delete thegene for TGFα showed a reduction in neuronal progenitor cells in thesubependyma and a reduction in neuronal progenitors that migrate to theolifactory bulb. In vitro, TGFα promoted dopamine uptake in fetal ratdopaminergic neurons in a dose-dependent and time-dependent manner. TGFαselectively promoted dopaminergic cell survival, enhanced neuritelength, branch number and the soma area of tyrosine hydroxylaseimmunopositive cells. The levels of TGFα were elevated in ventricularcerebrospinal fluid in juvenile parkinsonism and Parkinson's Disease andmay represent a compensatory response to neurodegeneration. Further,TGFα prevented a striatal neuronal degeneration in an animal model ofHuntington's Disease.

Nucleic Acids and Vectors

[0031] Polynucleotide or nucleic acid sequence refers to a polymericform of nucleotides. In some instances a polynucleotide refers to asequence that is not immediately contiguous with either of the codingsequences with which it is immediately contiguous (one on the 5′end andone on the 3′end) in the naturally occurring genome of the organism fromwhich it is derived. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector; into anautonomously replicating plasmid or virus; or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g., acDNA) independent of other sequences. The nucleotides of the inventioncan be ribonucleotides, deoxyribonucleotides, or modified forms ofeither nucleotide. In addition, the polynucleotide sequence involved inproducing a polypeptide chain can include regions preceding andfollowing the coding region (leader and trailer) as well as interveningsequences (introns) between individual coding segments (exons) dependingupon the source of the polynucleotide sequence.

[0032] The term polynucleotide(s) generally refers to anypolyribonucleotide or polydeoxyribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotidesas used herein refers to, among others, single-and double-stranded DNA,DNA that is a mixture of single- and double-stranded regions, single-and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded or a mixtureof single- and double-stranded regions.

[0033] In addition, the polynucleotides or nucleic acid sequences maycontain one or more modified bases. Thus, DNAs or RNAs with backbonesmodified for stability or for other reasons are “polynucleotides” asthat term is intended herein. Moreover, DNAs or RNAs comprising unusualbases, such as inosine, or modified bases, such as tritylated bases, toname just two examples, are polynucleotides as the term is used herein.

[0034] Nucleic acid sequences can be created which encode a fusionprotein (e.g., a TGF-α polypeptide and another polypeptide, such as atargeting sequence) and can be operatively linked to expression controlsequences. “Operatively linked” refers to ajuxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. For example, a coding sequence is“operably linked” to another coding sequence when RNA polymerase willtranscribe the two coding sequences into a single mRNA, which is thentranslated into a single polypeptide having amino acids derived fromboth coding sequences. The coding sequences need not be contiguous toone another so long as the expressed sequences ultimately process toproduce the desired protein. An expression control sequence operativelylinked to a coding sequence is ligated such that expression of thecoding sequence is achieved under conditions compatible with theexpression control sequences. As used herein, the term “expressioncontrol sequences” refers to nucleic acid sequences that regulate theexpression of a nucleic acid sequence to which it is operatively linked.Expression control sequences are operatively linked to a nucleic acidsequence when the expression control sequences control and regulate thetranscription and, as appropriate, translation of the nucleic acidsequence. Thus, expression control sequences can include appropriatepromoters, enhancers, transcription terminators, a start codon (ie.,ATG) in front of a protein-encoding gene, splicing signals for introns,maintenance of the correct reading frame of that gene to permit propertranslation of the mRNA, and stop codons. The term “control sequences”is intended to include, at a minimnm, components whose presence caninfluence expression, and can also include additional components whosepresence is advantageous, for example, leader sequences and fusionpartner sequences. Expression control sequences can include a promoter.

[0035] By “promoter” is meant minimal sequence sufficient to directtranscription. Also included in the invention are those promoterelements which are sufficient to render promoter-dependent geneexpression controllable for cell-type specific, tissue-specific, orinducible by external signals or agents; such elements may be located inthe 5′or 3′regions of the of a polynucleotide sequence. Bothconstitutive and inducible promoters, are included in the invention (seee.g., Bitter et al., Methods in Enzymology 153:516-544, 1987). Forexample, when cloning in bacterial systems, inducible promoters such aspL of bacteriophage, plac, ptrp, ptac (ptrp-lac hybrid promoter) and thelike may be used. When cloning in mammalian cell systems, promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the retrovirus long terminalrepeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter)may be used. Promoters produced by recombinant DNA or synthetictechniques may also be used to provide for transcription of the nucleicacid sequences of the invention.

[0036] A nucleic acid sequence of the invention including, for example,a polynucleotide encoding a fusion protein, may be inserted into arecombinant expression vector. A recombinant expression vector generallyrefers to a plasmid, virus or other vehicle known in the art that hasbeen manipulated by insertion or incorporation of a nucleic acidsequences. For example, a recombinant expression vector of the inventionincludes a polynucleotide sequence encoding a TGF-α polypeptide having asequence as set forth in SEQ ID NO:1, 2, 3, 4 or 6 or fragment thereof(as described more fully below). The expression vector typicallycontains an origin of replication, a promoter, as well as specific geneswhich allow phenotypic selection of the transformed cells. Vectorssuitable for use in the invention include, but are not limited to theT7-based expression vector for expression in bacteria (Rosenberg, etal., Gene 56:125, 1987), the pMSXND expression vector for expression inmammalian cells (Lee and Nathans, J. Biol. Chem. 263:3521, 1988),baculovirus-derived vectors for expression in insect cells, cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV. The nucleic acidsequences of the invention can also include a localization sequence todirect the indicator to particular cellular sites by fusion toappropriate organellar targeting signals or localized host proteins. Forexample, a polynucleotide encoding a localization sequence, or signalsequence, can be used as a repressor and thus can be ligated or fused atthe 5′terminus of a polynucleotide encoding a polypeptide or apolypeptide fragment of the invention such that the localization orsignal peptide is located at the amino terminal end of a resultingpolypeptide. The construction of expression vectors and the expressionof genes in transfected cells involves the use of molecular cloningtechniques also well known in the art. (See, for example, Sambrook etal., Molecular Cloning—A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1989, and Current Protocols inMolecular Biology, M. Ausubel et al., eds., (Current Protocols, a jointventure between Greene Publishing Associates, Inc. and John Wiley &Sons, Inc., most recent Supplement)). These methods include in vitrorecombinant DNA techniques, synthetic techniques and in vivorecombination/genetic recombination. (See also, Maniatis, et al.,Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory,N.Y., 1989).

[0037] In yeast, a number of vectors containing constitutive orinducible promoters may be used. For a review see, Current Protocols inMolecular Biology, Vol. 2, Ed. Ausubel, et al., Greene Publish. Assoc. &Wiley Interscience, Ch. 13, 1988; Grant, et al., “Expression andSecretion Vectors for Yeast,” in Methods in Enzymology, Eds. Wu &Grossman, 1987, Acad. Press, N.Y., Vol. 153, pp.516-544, 1987; Glover,DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch. 3, 1986; and Bitter,“Heterologous Gene Expression in Yeast,” Methods in Enzymology, Eds.Berger & Kimmel, Acad. Press, N.Y., Vol. 152, pp. 673-684, 1987; and TheMolecular Biology of the Yeast Saccharomyces, Eds. Strathern et al.,Cold Spring Harbor Press, Vols. I and II, 1982. A constitutive yeastpromoter such as ADH or LEU2 or an inducible promoter such as GAL may beused (“Cloning in Yeast,” Ch. 3, R. Rothstein In: DNA Cloning Vol. 11, APractical Approach, Ed. D M Glover, IRL Press, Wash., D.C., 1986).Alternatively, vectors may be used which promote integration of foreignDNA sequences into the yeast chromosome.

[0038] An alternative expression system which could be used to express aTGF-α polypeptide or function fragment of the invention is an insectsystem. In one such system, Autographa californica nuclear polyhedrosisvirus (AcNPV) is used as a vector to express foreign or mutatedpolynucleotide sequences. The virus grows in Spodoptera frugiperdacells. The sequence encoding a polypeptide of the invention may becloned into non-essential regions (for example, the polyhedrin gene) ofthe virus and placed under control of an AcNPV promoter (for example thepolyhedrin promoter). Successful insertion of the sequences coding for apolypeptide of the invention will result in inactivation of thepolyhedrin gene and production of non-occluded recombinant virus (i.e.,virus lacking the proteinaceous coat coded for by the polyhedrin gene).These recombinant viruses are then used to infect S. frugiperda cells inwhich the inserted gene is expressed, see Smith, et al., J. Viol.46:584, 1983; Smith, U.S. Pat. No. 4,215,051.

[0039] The vectors of the invention can be used to transform a hostcell. By transform or transformation is meant a permanent or transientgenetic change induced in a cell following incorporation of new DNA(i.e., DNA exogenous to the cell). Where the cell is a mammalian cell, apermanent genetic change is generally achieved by introduction of theDNA into the genome of the cell.

[0040] A transformed cell or host cell generally refers to a cell (e.g.,prokaryotic or eukaryotic) into which (or into an ancestor of which) hasbeen introduced, by means of recombinant DNA techniques, apolynucleotide molecule encoding a TGF-α polypeptide or functionalfragments thereof (e.g., a functional fragment as set forth in SEQ IDNO:4, as described below).

[0041] Transformation of a host cell with recombinant DNA may be carriedout by conventional techniques as are well known to those skilled in theart. Where the host is prokaryotic, such as E. coli, competent cellswhich are capable of DNA uptake can be prepared from cells harvestedafter exponential growth phase and subsequently treated by the CaCl₂method by procedures well known in the art. Alternatively, MgCl₂ or RbClcan be used. Transformation can also be performed after forming aprotoplast of the host cell or by electroporation.

[0042] When the host is a eukaryote, methods of transfection ortransformation with DNA include calcium phosphate co-precipitates,conventional mechanical procedures such as microinjection,electroporation, insertion of a plasmid encased in liposomes, or virusvectors, as well as others known in the art, may be used. Eukaryoticcells can also be cotransfected with DNA sequences encoding a TGF-αpolypeptide or fragment and a second foreign DNA molecule encoding aselectable marker, such as the herpes simplex thymidine kinase gene.Another method is to use a eukaryotic viral vector, such as simian virus40 (SV40) or bovine papilloma virus, to transiently infect or transformeukaryotic cells and express the protein. (Eukaryotic Viral Vectors,Cold Spring Harbor Laboratory, Gluzman ed., 1982). Typically, aeukaryotic host will be utilized as the host cell. The eukaryotic cellmay be a yeast cell (e.g., Saccharomyces cerevisiae), an insect cell(e.g., Drosophila sp.) or may be a mammalian cell, including a humancell.

[0043] Eukaryotic systems, and mammalian expression systems, allow forpost-translational modifications of expressed mammalian proteins tooccur. Eukaryotic cells which possess the cellular machinery forprocessing of the primary transcript, glycosylation, phosphorylation,and, advantageously secretion of the gene product should be used. Suchhost cell lines may include, but are not limited to, CHO, VERO, BHK,HeLa, COS, MDCK, Jurkat, HEK-293, and WI38.

[0044] Mammalian cell systems which utilize recombinant viruses or viralelements to direct expression may be engineered. For example, when usingadenovirus expression vectors, a polynucleotide encoding a TGF-αpolypeptide or fragment thereof may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric sequence may then be insertedin the adenovirus genome by in vitro or in vivo recombination. Insertionin a non-essential region of the viral genome (e.g., region E1 or E3)will result in a recombinant virus that is viable and capable ofexpressing a TGF-α polypeptide or fragment thereof in infected hosts(e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA, 81:3655-3659,1984). Alternatively, the vaccinia virus 7.5K promoter may be used.(e.g., see, Mackett, et al., Proc. Natl. Acad. Sci. USA, 79:7415-7419,1982; Mackett, et al., J. Virol. 49:857-864, 1984; Panicali, et al.,Proc. Natl. Acad. Sci. USA 79:4927-4931, 1982). Of particular interestare vectors based on bovine papilloma virus which have the ability toreplicate as extrachromosomal elements (Sarver, et al., Mol. Cell. Biol.1:486, 1981). Shortly after entry of this DNA into mouse cells, theplasmid replicates to about 100 to 200 copies per cell. Transcription ofthe inserted cDNA does not require integration of the plasmid into thehost's chromosome, thereby yielding a high level of expression. Thesevectors can be used for stable expression by including a selectablemarker in the plasmid, such as the neo gene. High level expression mayalso be achieved using inducible promoters, including, but not limitedto, the metallothionine IIA promoter and heat shock promoters.

[0045] For long-term, high-yield production of recombinant proteins,stable expression is preferred. Rather than using expression vectorswhich contain viral origins of replication, host cells can betransformed with the cDNA encoding a TGF-α polypeptide or functionalfragment controlled by appropriate expression control elements (e.g.,promoter, enhancer, sequences, transcription terminators,polyadenylation sites, etc.), and a selectable marker. The selectablemarker in the recombinant vector confers resistance to the selection andallows cells to stably integrate the plasmid into their chromosomes andgrow to form foci which in turn can be cloned and expanded into celllines. For example, following the introduction of foreign DNA,engineered cells may be allowed to grow for 1-2 days in an enrichedmedia, and then are switched to a selective media. A number of selectionsystems may be used, including, but not limited to, the herpes simplexvirus thymidine kinase (Wigler, et al, Cell, 11:223, 1977),hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski,Proc. Natl. Acad. Sci. USA, 48:2026, 1962), and adeninephosphoribosyltransferase (Lowy, et al., Cell, 22:817, 1980) genes canbe employed in tk-, hgprt- or aprt- cells respectively. Also,anti-metabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler, et al., Proc.Natl. Acad. Sci. USA, 77:3567, 1980; O'Hare, et al., Proc. Natl. Acad.Sci. USA, 8:1527, 1981); gpt, which confers resistance to mycophenolicacid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072, 1981); neo,which confers resistance to the aminoglycoside G-418 (Colberre-Garapin,et al., J. Mol. Biol. 150:1, 1981); and hygro, which confers resistanceto hygromycin (Santerre, et al., Gene 30:147, 1984) genes. Recently,additional selectable genes have been described, namely trpB, whichallows cells to utilize indole in place of tryptophan; hisD, whichallows cells to utilize histinol in place of histidine (Hartman &Mulligan, Proc. Natl. Acad. Sci. USA 85:8047, 1988); and ODC (ornithinedecarboxylase) which confers resistance to the ornithine decarboxylaseinhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue L., In:Current Communications in Molecular Biology, Cold Spring HarborLaboratory, ed., 1987).

[0046] The term “primer” as used herein refers to an oligonucleotide,whether natural or synthetic, which is capable of acting as a point ofinitiation of synthesis when placed under conditions in which primerextension is initiated or possible. Synthesis of a primer extensionproduct which is complementary to a nucleic acid strand is initiated inthe presence of nucleoside triphosphates and a polymerase in anappropriate buffer at a suitable temperature.

Proteins and Polypeptides

[0047] A polypeptide or protein refers to a polymer in which themonomers are amino acid residues which are joined together through amidebonds. When the amino acids are alpha-amino acids, either the L-opticalisomer or the D-optical isomer can be used, the L-isomers being typical.A TGF-α polypeptide or TGF-α related polypeptide is intended toencompass an amino acid sequence, including modified sequences such asglycoproteins, which exhibit TGF-α activity. The polypeptides of theinvention encompass amino acid sequences of human TGF-α as shown in SEQID NO:1 as well as polypeptides that have structural and/or functionalcharacteristics of TGF-α. For example, a polypeptide or a TGF-α relatedpolypeptide of the invention may include a polypeptide that shares acysteine disulfide bond structure similar to TGF-α such as a relatedfamily of proteins including vaccinia growth factor, amphiregulinprecursor, betacellulin precursor, heparin binding EGF-like growthfactor, epiregulin (rodent only), HUS 19878, myxomavirus growth factor(MGF), Shope fibroma virus growth factor (SFGF), and schwannoma derivedgrowth factor. In addition, a polypeptide of the invention will have oneor more functional characteristics related to TGF-α including, forexample, the ability to interact with an EGF family receptor member,stimulate proliferation or migration of stem cells, or to treat orprevent cachexia.

[0048] The polypeptides of the invention are intended to cover naturallyoccurring proteins, as well as those which are recombinantly orsynthetically synthesized. In addition, a TGF-α or related polypeptidecan occur in at least two different conformations wherein bothconformations have the same or substantially the same amino acidsequence but have different three dimensional structures so long as thehave a biological activity related to TGF-α . Polypeptide or proteinfragments of TGF-α are also encompassed by the invention such as thosedescribed by formulas I, II, and III (see below). Fragments can have thesame or substantially the same amino acid sequence as the naturallyoccurring protein. A polypeptide or peptide having substantially thesame sequence means that an amino acid sequence is largely, but notentirely, the same, but retains a functional activity of the sequence towhich it is related. In general polypeptides of the present inventioninclude peptides, or full length protein, that contains substitutions,deletions, or insertions into the protein backbone, that would stillhave an approximately 50%-70% homology to the original protein over thecorresponding portion. A yet greater degree of departure from homologyis allowed if like-amino acids, i.e. conservative amino acidsubstitutions, do not count as a change in the sequence. Polypeptidefragments of the invention retain a biological activity associated withTGF-α as described above.

[0049] Homology to TGF-α polypeptide can be measured using standardsequence analysis software (e.g., Sequence Analysis Software Package ofthe Genetics Computer Group, University of Wisconsin BiotechnologyCenter, 1710 University Avenue, Madison, Wis. 53705; also see Ausubel,et al., supra). Such procedures and algorithms include, for example, aBLAST program (Basic Local Alignment Search Tool at the National Centerfor Biological Information), ALIGN, AMAS (Analysis of Multiply AlignedSequences), AMPS (Protein Multiple Sequence Alignment), ASSET (AlignedSegment Statistical Evaluation Tool), BANDS, BESTSCOR, BIOSCAN(Biological Sequence Comparative Analysis Node), BLIMPS (BLocks IMProvedSearcher), FASTA, Intervals & Points, BMB, CLUSTAL V, CLUSTAL W,CONSENSUS, LCONSENSUS, WCONSENSUS, Smith-Waterman algorithm, DARWIN, LasVegas algorithm, FNAT (Forced Nucleotide Alignment Tool), Framealign,Framesearch, DYNAMIC, FILTER, FSAP (Fristensky Sequence AnalysisPackage), GAP (Global Alignment Program), GENAL, GIBBS, GenQuest, ISSC(Sensitive Sequence Comparison), LALIGN (Local Sequence Alignment), LCP(Local Content Program), MACAW (Multiple Alignment Construction &Analysis Workbench), MAP (Multiple Alignment Program), MBLKP, MBLKN,PIMA (Pattern-Induced Multi-sequence Alignment), SAGA (SequenceAlignment by Genetic Algorithm) and WHAT-IF.

[0050] A polypeptide may be substantially related but for a conservativevariation, such polypeptides being encompassed by the invention. Aconservative variation denotes the replacement of an amino acid residueby another, biologically similar residue. Examples of conservativevariations include the substitution of one hydrophobic residue such asisoleucine, valine, leucine or methionine for another, or thesubstitution of one polar residue for another, such as the substitutionof arginine for lysine, glutamic for aspartic acids, or glutamine forasparagine, and the like. Other illustrative examples of conservativesubstitutions include the changes of: alanine to serine; arginine tolysine; asparagine to glutamine or histidine; aspartate to glutamate;cysteine to serine; glutamine to asparagine; glutamate to aspartate;glycine to proline; histidine to asparagine or glutamine; isoleucine toleucine or valine; leucine to valine or isoleucine; lysine to arginine,glutamine, or glutamate; methionine to leucine or isoleucine;phenylalanine to tyrosine, leucine or methionine; serine to threonine;threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan orphenylalanine; valine to isoleucine to leucine. The term “conservativevariation” also includes the use of a substituted amino acid in place ofan unsubstituted parent amino acid provided that antibodies raised tothe substituted polypeptide also immunoreact with the unsubstitutedpolypeptide.

[0051] Modifications and substitutions are not limited to replacement ofamino acids. For a variety of purposes, such as increased stability,solubility, or configuration concerns, one skilled in the art willrecognize the need to introduce, (by deletion, replacement, or addition)other modifications. Examples of such other modifications includeincorporation of rare amino acids, dextra-amino acids, glycosylationsites, cytosine for specific disulfide bridge formation. The modifiedpeptides can be chemically synthesized, or the isolated gene can besite-directed mutagenized, or a synthetic gene can be synthesized andexpressed in bacteria, yeast, baculovirus, tissue culture and so on.

[0052] Solid-phase chemical peptide synthesis methods can also be usedto synthesize the polypeptide or fragments of the invention. Such methodhave been known in the art since the early 1960's (Merrifield, R. B., J.Am. Chem. Soc., 85, 2149-2154 (1963) (See also Stewart, J. M. and Young,J. D., Solid Phase Peptide Synthesis, 2 ed., Pierce Chemical Co.,Rockford, Ill., pp. 11-12)) and have recently been employed incommercially available laboratory peptide design and synthesis kits(Cambridge Research Biochemicals). Such commercially availablelaboratory kits have generally utilized the teachings of H. M. Geysen etal, Proc. Natl. Acad. Sci., USA, 81, 3998 (1984) and provide forsynthesizing peptides upon the tips of a multitude of “rods” or “pins”all of which are connected to a single plate. When such a system isutilized, a plate of rods or pins is inverted and inserted into a secondplate of corresponding wells or reservoirs, which contain solutions forattaching or anchoring an appropriate amino acid to the pin's or rod'stips. By repeating such a process step, i.e., inverting and insertingthe rod's and pin's tips into appropriate solutions, amino acids arebuilt into desired peptides. In addition, a number of available FMOCpeptide synthesis systems are available. For example, assembly of apolypeptide or fragment can be carried out on a solid support using anApplied Biosystems, Inc. Model 431A automated peptide synthesizer. Forexample, if the peptide is from formula I or formula II (see below), apreferred means for synthesizing peptides of 10-18 amino acids in lengthis by direct peptide synthesis generally starting with the N-terminalamino acid and adding amino acids in the C terminal direction. TGFα hasbeen made using recombinant techniques and is available as a laboratoryreagent commercially. The bifunctional compounds of formula III are bestsynthesized with each loop peptide moiety synthesized and then added tothe heterocyclic nitrogen atom using standard heterocyclic additionsynthesis.

TGF-α Peptide Mimics

[0053] The functional peptides of the invention are based upon thediscovery that a loop peptide of TGF-α exhibits TGF-α biologicalactivity and can therefore stimulate CNS multipotent precursor cells todivide and migrate through the brain. This activity indicates that theloop peptide is effective to treat neurological deficits caused by awide variety of diseases and injuries that each result in a neurologicaldeficit in some specific area of the brain or specific kind of neuron.These include degenerative diseases, including the more commonAlzheimer's Disease (AD), Parkinson's Disease (PD), and Huntington'sDisease (HD), and the less common Pick's disease, progressivesupranuclear palsy, striatonigral degeneration, cortico-basaldegeneration, olivopontocerebellar atrophy, Leigh's disease, infantilenecrotizing encephalomyelopathy, Hunter's disease,mucopolysaccharidosis, various leukodystrophies (such as Krabbe'sdisease, Pelizaeus-Merzbacher disease and the like), amaurotic(familial) idiocy, Kuf's disease, Spielmayer-Vogt disease, Tay Sachsdisease, Batten disease, Jansky-Bielschowsky disease, Reye's disease,cerebral ataxia, chronic alcoholism, beriberi, Hallervorden-Spatzsyndrome, cerebellar degeneration, and the like.

[0054] Further, injuries (traumatic or neurotoxic) that cause a loss ofneuronal function can be treated by the functional peptides. Suchinjuries include, for example, gunshot wounds, injuries caused by bluntforce, penetration injuries, injuries caused by surgical procedure(e.g., tumor removal, abscess removal, epilepsy lesion removal)poisoning (e.g., carbon monoxide), shaken baby syndrome, adversereactions to medications, drug overdoses, and post-traumaticencephalopathy. Ischemia can further cause CNS injury due to disruptionof blood flow or oxygen delivery that can kill or injure neurons andglial cells (e.g., TGF-α confers protection from ischemia in a porcinegastrointestinal model and a family member, Heparin-binding EGF, confersprotection from ischemia in a rat stroke model). Such injuries can betreated by administration of the functional peptides and include, forexample, injuries caused by stroke, anoxia, hypoxia, partial drowning,myoclonus, severe smoke inhalation, dystonias, and acquiredhydrocephalus. Developmental disorders that can be treated by thefunctional peptides include, for example, schizophrenia, certain formsof severe mental retardation, cerebral palsey, congenital hydrocephalus,severe autism, Downs Syndrome, LHRH/hypothalamic disorder, and spinabifida. The functional peptides can be further used to treat disordersaffecting vision caused by the loss or failure of retinal cells andinclude, for example, diabetic retinopathy, serious retinal detachment(associated with glaucoma), traumatic injury to the retina, retinalvascular occlusion, macular degeneration, optic nerve atrophy and otherretinal degenerative diseases. Injuries to the spinal cord can betreated by the functional peptides. Examples of spinal cord injuries arepost-polio syndrome, amyotrophic lateral sclerosis, traumatic injury,surgical injury, and paralytic diseases. Demylinating autoimmunedisorders can be treated by administration of the functional peptidesand include, for example, multiple sclerosis. Lastly, the functionalpeptides can be used to treat neurological deficits caused by infectionof inflammatory diseases, including, for example, Creutzfeldt-Jacobdisease and other slow virus infectious diseases of the CNS, AIDSencephalopathy, post-encephalitic Parkinsonism, viral encephalitis,bacterial meningitis and other CNS effects of infectious diseases.

[0055] By “functional” as used in connection with the peptides orpeptide fragments of the invention is meant that the peptides orfragments have TGFα activity. This biological activity is associatedwith the peptides of formula I, formula II and formula III and the dataavailable for TGFα.

[0056] Generally, the terms “treating”, “treatment” and the like areused herein to mean affecting a subject, tissue or cell to obtain adesired pharmacologic and/or physiologic effect. The effect may beprophylactic in terms of completely or partially preventing a disease ordisorder or sign or symptom thereof, and/or may be therapeutic in termsof a partial or complete cure for a disorder or disease and/or adverseeffect attributable to the disorder or disease. “Treating” as usedherein covers any treatment of, or prevention of, or inhibition of adisorder or disease in a subject. The subject can be an invertebrate, avertebrate, a mammal, and particularly a human, and includes by way ofexample: (a) preventing the disease or disorder from occurring in asubject that may be predisposed to the disease or disorder, but has notyet been diagnosed as having it; (b) inhibiting the disease or disorder,i.e., arresting its progression; or (c) relieving or ameliorating thedisease or disorder, i.e., causing regression.

[0057] The invention also provides methods of modulating weight-lossassociated with disease and disorders of the gastrointestinal tract, forexample, those associated with viral infections and chemotherapy byadministering TGF-α or related polypeptides or fragments thereof whichretain TGF-α biological activity (e.g., SEQ ID NO:1, 2, or 3, and thepeptides of formula I, II, or III).

[0058] The invention provides a peptide having TGFα biological activity,comprising at least an 11-membered peptide compound of formula I (SEQ IDNO:4):

NH₂-X_(1a)-Cys-His-Ser-X_(1b)-X₂-X_(1a)-X_(1b)-X_(1a)-X₃-Cys COOH   (I)

[0059] wherein X₁ is independently Val, Gly or Ala, wherein X₂ is Tyr orPhe, wherein X₃ is Arg or Lys, and wherein the two Cys moieties form adisulfide bond to create an 11-amino acid functional peptide having a 10member loop structure. In addition, at least one or more of thefollowing amino acids of formula II (SEQ ID NO:5) may be added to the Cterminus Cys moiety of formula I (SEQ ID NO:4):

-X₄-His-X_(1c)-X₄-X₅-X₆-X_(1c)   (II)

[0060] wherein X₄ is Glu or Asp, wherein X₅ is Leu or Ile, and whereinX₆ is Asp or Glu. Preferably, X_(1a) is Val, X_(1b) is Gly and X_(1c) isAla thereby producing an 11, 12, 13, 14, 15, 16, 17 or 18 amino acidpeptide. Preferably, X₂ is Tyr, and X₃ is Arg. Accordingly, in oneembodiment the functional peptide of the invention has a sequence:NH₂-X_(1a)-Cys-His-Ser-X_(1b)-X₂-X_(1a)-X_(1b)-X_(1a)-X₃-Cys-X₄-His-X_(1c)-X₄-X₅-X₆-X_(1c)-COOH(SEQ ID NO:6)

[0061] SEQ ID NO:6 forms a 10 member loop structure with a 7 member tailthat can be varied in length. In addition, SEQ ID NO:6 can form dimerscomprising, for example, a 34-mer peptide. Accordingly, the functionalpeptide can be from about 10 to 18 amino acids in length (e.g., 10, 11,12, 13, 14, 15, 16, 17, or 18 amino acids) wherein X_(1a) is Val, X_(1b)is Gly, X_(1c), is Ala and X₄ is Gly and may also comprise hetero- orhomo-dimers of various TGF-α peptides described herein. Such dimers mayhave greater or reduced activities as compared to monomers.

[0062] The invention further provides a pharmaceutical compositioncomprising a peptide in a pharmaceutically acceptable carrier, whereinthe peptide compound comprises at least about a 10 to 18-memberedpeptide compound of formula I (SEQ ID NO:4, including members of SEQ IDNO:5 attached to SEQ ID NO:4 and including SEQ ID NO:6). Preferably, atleast one or more of the seven amino acids of formula II are added tothe C terminus Cys moiety. Most preferably, the functional peptide is 18amino acids in length wherein X_(1a) is Val, X_(1b) is Gly, X_(1c) isAla and X₄ is Gly. The peptides described herein are all useful in themethods of the invention.

[0063] The invention further provides a method for treating aneurodegenerative disease with a pharmaceutically active TGF-αpolypeptide, functional fragment peptide thereof or a pharmaceuticallyactive TGF-α57 polypeptide, wherein the peptide comprises at least an11-membered peptide compound of formula I or a polypeptide of formulaIII, wherein formula I (SEQ ID NO:4) is:

NH₂-X_(1a)-Cys-His-Ser-X_(1b)-X₂-X_(1a)-X_(1b)-X_(1a)- X₃-Cys COOH   (I)

[0064] wherein X₁ is independently Val, Gly or Ala, wherein X₂ is Try orPhe, wherein X₃ is Arg or Lys, and wherein the two Cys moieties form adisulfide bond to create an 11-amino acid functional peptide; andwherein formula III is:

Loop peptide N-terminus-linker-cyclic C₄H₈N₂ -linker-Loop peptideN-terminus   (III)

[0065] wherein the linker moiety is designed to link the N-terminus ofthe Loop peptide to a nitrogen atom of the ring C₄H₈N₂ and wherein the“loop peptide” comprises at least an 11-membered peptide compound offormula I (SEQ ID NO:4); wherein X₁ is independently Val, Gly or Ala,wherein X₂ is Try or Phe, wherein X₃ is Arg or Lys, and wherein the twoCys moieties form a disulfide bond to create an 11-amino acid functionalpeptide having TGF-α activity. Furthermore, the functional peptides ofthe invention, as described above (e.g., SEQ ID Nos: 4, 5, and 6),having from about 10 to 18 amino acids in length (e.g., 10, 11, 12, 13,14, 15, 16, 17, or 18 amino acids) wherein X_(1a) is Val, X_(1b) is Gly,X_(1c) is Ala and X₄ is Gly and hetero- or homo-dimers of the variousTGF-α peptides described herein can be used in the methods of theinvention.

[0066] The invention further provides a method for treating aneurodegenerative disease with an pharmaceutically active TGF-α57polypeptide (SEQ ID NO:3), wherein TGF-α57 is a 57 amino acidpolypeptide having the formula IV:

Ser-Leu-Ser-Leu-Pro-Ala-Met-Human TGFα  (IV)

[0067] wherein human TGFα is a 50 amino acid polypeptide having asequence as set forth in SEQ ID NO:1.

[0068] The invention further provides a method for treating a CNSdisease or disorder, wherein the CNS disease or disorder includes CNSischemia, spinal cord injury, MS, and retinal injury, with apharmaceutically active TGFα peptide or a TGFα57 polypeptide, whereinthe peptide comprises at least an 11-membered peptide compound offormula I (SEQ ID NO:4):

NH₂-X_(1a)-Cys-His-Ser-X_(1b)-X₂-X_(1a)-X_(1b)-X_(1a)-X₃-Cys-COOH   (I)

[0069] wherein X₁ is independently Val, Gly or Ala, wherein X₂ is Try orPhe, wherein X₃ is Arg or Lys, and wherein the two Cys moieties form adisulfide bond to create an 11-amino acid functional TGF-α peptide; andwherein TGF-α57 is a 57 amino acid polypeptide having the formula IV:

Ser-Leu-Ser-Leu-Pro-Ala-Met-Human TGFα  (I)

[0070] wherein human TGFα is a 50 amino acid polypeptide having theformula of SEQ ID NO:1. Preferably, at least one or more of thefollowing amino acids from formula II are added to the C terminus Cysmoiety of formula I:

-X₄-His-X_(1c)-X₄-X₅-X₆-X_(1c)   (II)

[0071] wherein X₄ is Glu or Asp, wherein X₅ is Leu or Ile, and whereinX₆ is Asp or Glu. Preferably, X_(1a) is Val, X_(1b) is Gly and X_(1c) isAla. Preferably, X₂ is Tyr, and X₃ is Arg. Most preferably, thefunctional peptide is 18 amino acids in length wherein X_(1a) is Val,X_(1b) is Gly, X_(1c) is Ala and X₄ is Gly. Furthermore, the functionalpeptides of the invention, as described above (e.g., SEQ ID Nos:4, 5,and 6), having from about 10 to 18 amino acids in length (e.g., 10, 11,12, 13, 14, 15, 16, 17, or 18 amino acids) wherein X_(1a) is Val, X_(1b)is Gly, X_(1c) is Ala and X₄ is Gly and hetero- or homo-dimers of thevarious TGF-α peptides described herein can be used in the methods fortreating a CNS disease or disorder.

[0072] The invention further provides a method for enhancinghematopoiesis and myelopoiesis during cytotoxic or immune-suppressingtherapy, comprising administering a TGFα polypeptide (SEQ ID NO:1), aTGFα57 polypeptide (i.e., formula IV), a functional TGF-α peptidethereof, or a combination thereof, wherein the peptide comprises atleast an 11-membered peptide compound of formula I (SEQ ID NO:4):

NH₂-X_(1a)-Cys-His-Ser-X_(1b)-X₂-X_(1a)-X_(1b)-X_(1a)-X₃-Cys COOH   (I)

[0073] wherein X₁ is independently Val, Gly or Ala, wherein X₂ is Try orPhe, wherein X₃ is Arg or Lys, and wherein the two Cys moieties form adisulfide bond to create an 11-amino acid functional TGF-α peptide.Preferably, at least one or more of the following amino acids of formulaII are added to the C terminus Cys moiety of formula I:

-X₄-His-X_(1c)-X₄-X₅-X₆-X_(1c)   (II)

[0074] wherein X₄ is Glu or Asp, wherein X₅ is Leu or Ile, and whereinX₆ is Asp or Glu. Preferably, X_(1a) is Val, X_(1b) is Gly and X_(1c) isAla. Preferably, X₂ is Tyr, and X₃ is Arg. Most preferably, thefunctional peptide is 18 amino acids in length wherein X_(1a) is Val,X_(1b) is Gly, X_(1c) is Ala and X₄ is Gly. Furthermore, the functionalpeptides of the invention, as described above (e.g., SEQ ID Nos:4, 5,and 6), having from about 10 to 18 amino acids in length (e.g., 10, 11,12, 13, 14, 15, 16, 17, or 18 amino acids) wherein X_(1a) is Val, X_(1b)is Gly, X_(1c) is Ala and X₄ is Gly and hetero- or homo-dimers of thevarious TGF-α peptides described herein can be used in the methods forenhancing hematopoiesis or myelopoiesis. Preferably, the inventionfurther comprises administering a second hematopoietic growth factoragent to stimulate more mature hematopoietic precursor cells, whereinthe second hematopoietic growth factor includes erythropoietin,thrombopoietin, G-CSF (granulocyte colony stimulating factor), andGM-CSF (granulocyte macrophage colony stimulating factor).

[0075] The invention further provides a method for treating orpreventing mucositis of the gastrointestinal tract caused by cytotoxicor immune-suppressing therapy, comprising administering a TGF-αpolypeptide (SEQ ID NO:1), a TGF-α57 polypeptide (i.e., formula IV), afunctional TGF-α peptide thereof, or combinations thereof, wherein thepeptide comprises at least an 11-membered peptide compound of formula I(SEQ ID NO:4):

NH₂-X₁-Cys-His-Ser-X_(1b)-X₂-X_(1a)-X_(1b)-X_(1a)-X₃-Cys COOH   (I)

[0076] wherein X_(1a) and X_(1b) are independently Val, Gly or Ala; X₂is Tyr or Phe; X₃ is Arg or Lys; and the two Cys moieties are linked viaa disulfide bond to form an at least 11-amino acid functional peptidehaving TGF-α activity. Preferably, at least one or more of the followingamino acids are added to the C terminus Cys moiety from formula II:

-X₄-His-X_(1c)-X₄-X₅-X₆-X_(1c)   (II)

[0077] wherein X₄ is Glu or Asp, wherein X₅ is Leu or Ile, and whereinX₆ is Asp or Glu. Preferably, X_(1a) is Val, X_(1b) is Gly and X_(1c) isAla. Preferably, X₂ is Tyr, and X₃ is Arg. Most preferably, thefunctional peptide is 18 amino acids in length wherein X_(1a) is Val,X_(1b) is Gly, X_(1c) is Ala and X₄ is Gly. Furthermore, the functionalpeptides of the invention, as described above (e.g., SEQ ID Nos:4, 5,and 6), having from about 10 to 18 amino acids in length (e.g., 10, 11,12, 13, 14, 15, 16, 17, or 18 amino acids) wherein X_(1a) is Val, X_(1b)is Gly, X_(1c) is Ala and X₄ is Gly and hetero- or homo-dimers of thevarious TGF-α peptides described herein can be used in the methods fortreating or preventing mucositis.

[0078] The invention further provides a bifunctional compound that actsas a TGFα mimetic, comprising a compound of formula III:

Loop peptide N-terminus-linker-cyclic C₄H₈N₂-linker-Loop peptideN-terminus   (III)

[0079] wherein the linker moiety is designed to link the N-terminus ofthe Loop peptide to a nitrogen atom of the ring C₄H₈N₂ and wherein the“loop peptide” comprises at least an 11-membered peptide compound offormula I:

NH₂-X_(1a)-Cys-His-Ser-X_(1b)-X₂-X_(1a)-X_(1b)-X_(1a)-X₃-Cys COOH   (I)

[0080] wherein X_(1a) and X_(1b) are independently Val, Gly or Ala; X₂is Tyr or Phe; X₃ is Arg or Lys; and the two Cys moieties are linked viaa disulfide bond to form an at least 11-amino acid functional peptidehaving TGF-α activity. Preferably, at least one or more of the followingamino acids are added to the C terminus Cys moiety from formula II:

-X₄-His-X_(1c)-X₄-X₅ -X₆-X_(1c)   (II)

[0081] wherein X₄ is Glu or Asp, wherein X₅ is Leu or Ile, and whereinX₆ is Asp or Glu. Preferably, X_(1a) is Val, X_(1b) is Gly and X_(1c) isAla. Preferably, the linker group is independently selected from thegroup consisting of substituted or unsubstituted C₁₋₆ alkyl, substitutedor unsubstituted C₂₋₆ alkenyl, substituted or unsubstituted C₁₋₆ alkoxy,xylenyl, wherein the substitutions are selected from the groupconsisting of oxo, epoxyl, hydroxyl, chloryl, bromyl, fluoryl, andamino. Preferably, X₂ is Tyr, and X₃ is Arg. Most preferably, thefunctional peptide is 18 amino acids in length wherein X_(1a) is Val,X_(1b) is Gly, X_(1c) is Ala and X₄ is Gly.

[0082] The invention further provides a method for treating inflammatorybowel disease, colitis, and Chron's Disease of the gastrointestinaltract, comprising administering a TGF-α polypeptide (SEQ ID NO:1), aTGF-α57 polypeptide (formula IV), a functional TGF-α peptide thereof, orcombinations thereof, wherein the peptide comprises at least an11-membered peptide compound of formula I:

NH₂-X_(1a)-Cys-His-Ser-X_(1b)-X₂-X_(1a)-X_(1b)-X_(1a)-X₃-Cys COOH   (I)

[0083] wherein X₁ is independently Val, Gly or Ala, wherein X₂ is Try orPhe, wherein X₃ is Arg or Lys, and wherein the two Cys moieties form adisulfide bond to create an 11-amino acid functional TGF-α peptide.Preferably, at least one or more of the following amino acids are addedto the C terminus Cys moiety from formula II:

-X₄-His-X_(1c)-X₄-X₅-X₆-X_(1c)   (II)

[0084] wherein X₄ is Glu or Asp, wherein X₅ is Leu or Ile, and whereinX₆ is Asp or Glu. Preferably, X_(1a) is Val, X_(1b) is Gly and X_(1c) isAla. Preferably, X₂ is Tyr, and X₃ is Arg. Most preferably, thefunctional peptide is 18 amino acids in length wherein X_(1a) is Val,X_(1b) is Gly, X_(1c) is Ala and X₄ is Gly. Furthermore, the functionalpeptides of the invention, as described above (e.g., SEQ ID Nos:4, 5,and 6), having from about 10 to 18 amino acids in length (e.g., 10, 11,12, 13, 14, 15, 16, 17, or 18 amino acids) wherein X_(1a) is Val, X_(1b)is Gly, X_(1c) is Ala and X₄ is Gly and hetero- or homo-dimers of thevarious TGF-α peptides described herein can be used in the methods fortreating a inflammatory bowel disease, colitis, Chron's Disease and thelike.

[0085] The invention further provides a method for treating aninflammatory reaction or autoimmune diseases resulting in weight-loss,comprising administering a TGFα polypeptide (SEQ ID NO:1), a TGFα57polypeptide (formula IV), a functional TGF-α peptide thereof, orcombinations thereof, wherein the peptide comprises at least an11-membered peptide compound of formula I:

NH₂-X_(1a)-Cys-His-Ser-X_(1b)-X₂-X_(1a)-X_(1b)-X_(1a)-X₃-Cys COOH   (I)

[0086] wherein X₁ is independently Val, Gly or Ala, wherein X₂ is Try orPhe, wherein X₃ is Arg or Lys, and wherein the two Cys moieties form adisulfide bond to create an 11-amino acid functional TGF-α peptide.Preferably, at least one or more of the following amino acids are addedto the C terminus Cys moiety from formula II (SEQ ID NO:5):

-X₄-His-X_(1c)-X₄-X₅-X₆-X_(1c)   (II)

[0087] wherein X₄ is Glu or Asp, wherein X₅ is Leu or Ile, and whereinX₆ is Asp or Glu. Preferably, X_(1a) is Val, X_(1b) is Gly and X_(1c) isAla. Preferably, X₂ is Tyr, and X₃ is Arg. Most preferably, thefunctional peptide is 18 amino acids in length wherein X_(1a) is Val,X_(1b) is Gly, X_(1c) is Ala and X₄ is Gly. Furthermore, the functionalpeptides of the invention, as described above (e.g., SEQ ID Nos:4, 5,and 6), having from about 10 to 18 amino acids in length (e.g., 10, 11,12, 13, 14, 15, 16, 17, or 18 amino acids) wherein X_(1a) is Val, X_(1b)is Gly, X_(1c) is Ala and X₄ is Gly and hetero- or homo-dimers of thevarious TGF-α peptides described herein can be used in the methods fortreating an autoimmune disease or inflammatory disorder. Preferably, theautoimmune diseases includes Type II (Juvenile) Diabetes, rheumatoidarthritis, lupus, HIV-associated disorders (e.g., AIDS) and multiplesclerosis.

Gene Therapy and Gene Delivery

[0088] The TGF-α polypeptides (e.g., SEQ ID NO:1), TGF-α57 polypeptide,and functional TGF-α peptides thereof are particularly suited fordelivery to a subject by means of a nucleic acid gene expression systemex vivo or in vivo. A variety of transfection techniques are currentlyavailable and used to transfer DNA in vitro into cells; includingcalcium phosphate-DNA precipitation, DEAE-Dextran transfection,electroporation, liposome mediated DNA transfer or transduction withrecombinant viral vectors. Such ex vivo treatment protocols have beenused to transfer DNA into a variety of different cell types includingepithelial cells (U.S. Pat. No. 4,868,116; Morgan and MulliganWO87/00201; Morgan et al., 1987, Science 237:1476-1479; Morgan andMulligan, U.S. Pat. No. 4,980,286), endothelial cells (WO89/05345),hepatocytes (WO89/07136; Wolff et al., 1987, Proc. Natl. Acad. Sci. USA84:3344-3348; Ledley et al., 1987 Proc. Natl. Acad. Sci. 84:5335-5339;Wilson and Mulligan, WO89/07136; Wilson et al., 1990, Proc. Natl. Acad.Sci. 87:8437-8441) fibroblasts (Palmer et al., 1987, Proc. Nati. Acad.Sci. USA 84:1055-1059; Anson et al., 1987, Mol. Biol. Med. 4:11-20;Rosenberg et al., 1988, Science 242:1575-1578; Naughton & Naughton, U.S.Pat. No. 4,963,489), lymphocytes (Anderson et al., U.S. Pat. No.5,399,346; Blaese, R. M. et al., 1995, Science 270:475-480) andhematopoietic stem cells (Lim, B. et al. 1989, Proc. Natl. Acad. Sci.USA 86:8892-8896; Anderson et al., U.S. Pat. No. 5,399,346). A summaryof typical protocols, methodology, and vectors is provided in “TheDevelopment of Human Gene Therapy,” Ed. Theodore Friedmann, Cold SpringHarbor Laboratory Press, New York, 1999, the disclosure of which isincorporated herein.

[0089] Direct in vivo gene transfer has recently been attempted withformulations of DNA trapped in liposomes (Ledley et al., 1987, JPediatrics 110:1); or in proteoliposomes that contain viral envelopereceptor proteins (Nicolau et al., 1983, Proc. Natl. Acad. Sci. U.S.A.80:1068); and DNA coupled to a polylysine-glycoprotein carrier complex.In addition, “gene guns” have been used for gene delivery into cells(Australian Patent No. 9068389). Naked DNA, or DNA associated withliposomes, can be formulated in liquid carrier solutions for injectioninto interstitial spaces for transfer of DNA into cells (Felgner,WO90/11092).

[0090] As described above, polynucleotide sequences encoding a TGF-αpolypeptide or function peptide fragment, can be cloned into vectorssuitable for delivery to host cells for expression. In particularretroviral vectors containing the polypeptides of the invention areparticularly suitable for delivering polynucleotides to cells for genetherapy. Current strategies for gene therapy are reviewed in “TheDevelopment of Human Gene Therapy,” Ed. Theodore Friedmann, Cold SpringHarbor Laboratory Press, New York, 1999, the disclosure of which isincorporated herein.

[0091] Delivery of a polynucleotide of interest may be accomplished invivo by administration of the vectors to an individual subject,typically by systemic administration (e.g., intravenous,intraperitoneal, intramuscular, subdermal, or intracranial infusion).Alternatively, the vectors may be used to deliver polynucleotides tocells ex vivo such as cells explanted from an individual patient (e.g.,tumor-infiltrating lymphocytes, bone marrow aspirates, tissue biopsy) oruniversal donor hematopoietic stem cells, followed by reimplantation ofthe cells into a patient, usually after selection for cells which haveincorporated the polynucleotide.

[0092] The vectors may be used for gene therapy to reduce the incidenceof weight-loss and associated disorders resulting from particulardiseases (e.g., cancer), or viral diseases (e.g., AIDS, mononucleosis,herpesvirus infection, cytomegalovirus infection, papillomavirusinfection) or to modify the genome of selected types of cells of apatient for any therapeutic benefit.

[0093] The vectors of the invention can be used to introducepolynucleotides into a variety of cells and tissues including myeloidcells, bone marrow cells, lymphocytes, hepatocytes, fibroblasts, lungcells, epithelial cells and muscle cells. For example, polynucleotidesencoding a TGF-α polypeptide may be transferred to stem cells.

Pharmaceutical Composition and Formulations

[0094] The invention includes various pharmaceutical compositions usefulfor delivery or administration of the peptides of the invention. In oneembodiment the pharmaceutical composition are useful in treating orpreventing weight-loss associated with a disorder or disease. Suchdisorders or diseases include weight-loss attributable to, for example,chemotherapy or a viral infection (e.g., HIV). The pharmaceuticalcompositions according to the invention are prepared by bringing apolypeptide or peptide derivative of TGF-α, a TGF-α mimetic into a formsuitable for administration to a subject using carriers, excipients andadditives or auxiliaries. Frequently used carriers or auxiliariesinclude magnesium carbonate, titanium dioxide, lactose, mannitol andother sugars, talc, milk protein, gelatin, starch, vitamins, celluloseand its derivatives, animal and vegetable oils, polyethylene glycols andsolvents, such as sterile water, alcohols, glycerol and polyhydricalcohols. Intravenous vehicles include fluid and nutrient replenishers.Preservatives include antimicrobial, anti-oxidants, chelating agents andinert gases. Other pharmaceutically acceptable carriers include aqueoussolutions, non-toxic excipients, including salts, preservatives, buffersand the like, as described, for instance, in Remington's PharmaceuticalSciences, 15th ed. Easton: Mack Publishing Co., 1405-1412, 1461-1487(1975) and The National Formulary XIV., 14th ed. Washington: AmericanPharmaceutical Association (1975), the contents of which are herebyincorporated by reference. The pH and exact concentration of the variouscomponents of the pharmaceutical composition are adjusted according toroutine skills in the art. See Goodman and Gilman's The PharmacologicalBasis for Therapeutics (7th ed.).

[0095] The pharmaceutical compositions are preferably prepared andadministered in dose units. Solid dose units are tablets, capsules andsuppositories and including, for example, alginate based pH dependentrelease gel caps. For treatment of a subject, depending on activity ofthe compound, manner of administration, nature and severity of thedisorder, age and body weight of the subject, different daily doses arenecessary. Under certain circumstances, however, higher or lower dailydoses may be appropriate. The administration of the daily dose can becarried out both by single administration in the form of an individualdose unit or by several smaller dose units and also by multipleadministration of subdivided doses at specific intervals.

[0096] The pharmaceutical compositions according to the invention may beadministered locally or systemically in a therapeutically effectivedose. Amounts effective for this use will, of course, depend on theseverity of the disease and the weight and general state of the subject.Typically, dosages used in vitro may provide useful guidance in theamounts useful for in situ administration of the pharmaceuticalcomposition, and animal models may be used to determine effectivedosages for treatment of particular disorders. Various considerationsare described, e.g., in Langer, Science, 249: 1527, (1990); Gilman etal. (eds.) (1990), each of which is herein incorporated by reference.

[0097] In one embodiment, the invention provides a pharmaceuticalcomposition useful for administering a TGF-α polypeptide or functionalfragment, or a nucleic acid encoding a TGF-α polypeptide or functionalfragment, to a subject in need of such treatment. “Administering” thepharmaceutical composition of the invention may be accomplished by anymeans known to the skilled artisan. Preferably a “subject” refers to amammal, most preferably a human.

[0098] The TGF-α polypeptide or functional fragment can be administeredparenterally, enterically, by injection, rapid infusion, nasopharyngealabsorption, dermal absorption, rectally and orally. Pharmaceuticallyacceptable carrier preparations for parenteral administration includesterile or aqueous or non-aqueous solutions, suspensions, and emulsions.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Carriers for occlusive dressings can be used toincrease skin permeability and enhance antigen absorption. Liquid dosageforms for oral administration may generally comprise a liposome solutioncontaining the liquid dosage form. Suitable solid or liquidpharmaceutical preparation forms are, for example, granules, powders,tablets, coated tablets, (micro)capsules, suppositories, syrups,emulsions, suspensions, creams, aerosols, drops or injectable solutionin ampule form and also preparations with protracted release of activecompounds, in whose preparation excipients and additives and/orauxiliaries such as disintegrants, binders, coating agents, swellingagents, lubricants, flavorings, sweeteners and elixirs containing inertdiluents commonly used in the art, such as purified water. Where thedisease or disorder is a gastrointestinal disorder oral formulations orsuppository formulations are preferred.

[0099] Sterile injectable solutions can be prepared by incorporating theactive agent (see formula I, formula II, or formula III and TGFα) in therequired amount (e.g., about 10 μg to about 10 mg/kg) in an appropriatesolvent and then sterilizing, such as by sterile filtration. Further,powders can be prepared by standard techniques such as freeze drying orvacuum drying.

[0100] In another embodiment, the active agent is prepared with abiodegradable carrier for sustained release characteristics for eithersustained release in the GI tract or for target organ implantation withlong term active agent release characteristics to the intended site ofactivity. Biodegradable polymers include, for example, ethylene vinylacetate, polyanhydrides, polyglycolic acids, polylactic acids, collagen,polyorthoesters, and poly acetic acid. Liposomal formulation can also beused.

[0101] The invention will now be described in greater detail byreference to the following non-limiting examples.

EXAMPLE 1

[0102] Each of the three loop regions in human TGFα was investigated forTGFα-like biological activity, such as stimulation of cellularproliferation as measured by ³H thymidine incorporation of stem cells.As shown in FIG. 2, only the Loop C peptide (corresponding to aminoacids 33-50) showed significant TGF-α biological activity as compared todata obtained with TGF-α 50 amino acid polypeptide or even the alteredsplice 57 amino acid polypeptide and is therefore a TGF-α mimeticpeptide. Accordingly, data from TGFα or TGF-α57 show what can be called“TGF-α activity” and that these are predictive of activity of thefunctional TGF-α peptide and similar functional TGF-α peptides embodiedin the genus of formula I with or without the addition of a “tail”region of formula II. These data predict activity for the functionalTGF-α peptides when activity is also shown for TGF-α or for TGF-α57.

EXAMPLE 2 Hematopoiesis

[0103] TGF-α and related polypeptides, such as TGF-α57, showedsurprising enhancing activity in an in vivo model of generalhematopoiesis when administered in conjunction with a potent cytotoxicagent Cis Platinum (CP). FIG. 3 shows a graph of mouse spleen weightsthat were treated with CP at either 5 μg/g or 10 μg/g and with TGF-α57at concentrations of 10 ng/g or 50 ng/g. These data show that TGF-α57treatment caused a return to normal spleen weights despite CP treatmentthat reduced spleen weights significantly. This in vivo experiment is apredictive model for hematopoiesis in humans as CP is a cytotoxic agentcommonly used for cancer chemotherapy that is known to significantlyreduce trilineage hematopoietic cells. Hematopoietic and myeloid cellsare red blood cell precursors, platelet precursors (megakaryocytes), andimmune (white) blood cell precursors of various forms of T cells, Bcells and macrophages. Moreover, platelet counts were higher in thosemice injected with TGF-α57 (and CP) as opposed to CP alone were suchcounts were significantly reduced from normal. It should be noted thatreferences to TGF-α as a human 50 amino acid polypeptide further includereference to human TGFα57 as an alternative variant.

[0104] The experiment procedure dosed those animals to be treated withTGFα57 4 hours prior to challenge with CP. A single dose of CP wasadministered. Additional doses (as indicated) of TGFα57 were made at 24hours, 48 hours, 72 hours and 96 hours after the CP dose. All doses weremade by IP injection. Controls were dosed with saline instead of eitheror both of CP and TGF-α57.

[0105] The animals were sacrificed about 4 hours after the last TGF-α57(or saline) dose. Key organs were removed and spleens were immediatelyweighed after a clean incision. All the relevant organs were placed informalin, transported for histopathological analysis, mounted,sectioned, stained and observed. The results of this histologicalanalysis of the spleens for hematopoietic effect and the GI tract(below) provided surprising and unexpected data of the effect of TGF-α57activity.

[0106] H&E-stained spleens of a CP-treated mouse spleen (10 μg/g) showedapoptotic cells (densely stained with fragments of nuclei) in thegerminal center (GC). T cells in the central arterial area showed theabsence of a marginal zone and much fewer erythrocytes and T cells inthe perifolecular area. A normal mouse spleen (no CP and no TGF-α57)fixed in formalin showed an arteriole enriched for T cell progenitors.There was erythrocytes in the perifollicular zone surrounding both the Tcell and B cell compartments of white pulp. A mouse spleen treated withCP (10 μg/g) and TGF-α57 (50 ng/g) showed an increased number of T cellsand erythrocytes in the perifollicular zone. The T cells stained for theT-cell receptor but were negative for CD4 and CD8 markers. Accordingly,the T-cells are double null T-cell progenitors induced by TGF-αadministration.

[0107] These in vivo data in a predictive model of hematopoiesis andconfirmed by blinded histological analysis (the histologist/pathologistwas blinded as to the treatment history of the coded tissues received)providing surprising evidence of the utility of peptides having TGF-αactivity to augment hematopoiesis and genesis of immune cells followingcytotoxic exposure. These data predict and provide a reasonablecorrelation that TGF-α and the peptides of formula I, formula II andformula III are useful therapeutic agents for enhancing hematopoiesisfollowing or during cytotoxic therapy, such as cancer treatment.Therefore, a useful method for improving cancer chemotherapy is tocombine either TGF-α or a peptide from formula I, formula II, formulaIII, of formula IV or combinations thereof with cytotoxic treatmentregimens to reduce dose-limiting side effects of cytotoxic agents.

[0108] An additional experiment investigated TGF-α activity (usingTGF-α57) on human bone-marrow enriched CD34 cells. FACS-sorted humanCD34 positive and CD38 negative cells were cultured in liquid primarycultures in Iscove's modified Dulbecco's media with supplements. TGFα(57) was added alone (10 ng/ml) and exhibited a 35% increase in CD34positive progenitor cells. Stem Cell Factor (SCF) was used as a positivecontrol (500 ng/ml) and provided a three-fold increase in CD34 positivecells. When a combination of SCF (500 ng/ml) and TGFα (10 ng/ml) wasadded, a synergistic 12-fold increase in CD34 positive cells wasobserved. An unexpected result was the stimulation of the proliferationof dendritic precurso cells in the TGF-α treated cultures.

EXAMPLE 3 Mucositis and Gastrointestinal Diseases

[0109] The small intestine comprises the duodenum, jejunum and ileum. Itis the principal site for absorption of digestive products from the GItract. Digestion begins in the stomach and is completed in the smallintestine in association with the absorptive process. The intestinalmucosa surface is made up of numerous finger-like projections calledvilli. In addition, mucosal epithelium between the basis of the villi isformed into the crypts which contain stem cells.

[0110] TGFα or a peptide from formula I, formula II, formula III, orformula IV having TGFα activity or combinations thereof are also usefulfor treating mucositis associated intestinal bleeding, dyspepsia causedby with cytotoxic therapy and for improving the barrier function of theGI tract compromised by cytotoxic therapy. The in vivo experiment withseven groups of mice described above for hematopoietic effects noted inspleens also examined the GI tract of these treated mice. Histologicalexamination of mouse intestines showed the following: CP (single ip doseof 10 μg/g) treated intestine, when cross-sectioned, showed significantinjury to the villi. Specifically, the villi are necrotic, the cryptsare in irregular shapes, and the tips of the crypts were exhibiting lossof cellular integrity. A cross section of a normal mouse GI tract (no CPand no TGFα57) showed a normal intestinal surface with villi having longand slender mucosal projections with a core of lamina propria covered bya luminal epithelial layer. A single row of intestinal crypt is found atthe base of the mucosa. These crypts that lie between adjacent villi aresurrounded by the same lamina propria that form the villous cores. Bothcolumnar absorptive cells and goblet cells cover the villous surfaces.The goblet cells contain apical clear vacuoles. A cross section of amouse intestine exposed to both the CP (10 μg/g) and TGFα57 (50 ng/g)showed that the intestinal structure was very similar to the normalintestinal structure. Specifically, the villus was long and slender.Both absorptive cells and goblet cells were visible at the surface ofthe villi, and there was an abundant amount of goblet cells on thesurface.

[0111] A 160× magnification of the intestines of a CP-treated mouse, anormal mouse and a CP treated and TGFα57 treated mouse at the same dosesas described above. The CP-treated mouse showed injured villi withdegenerating and necrotic tips. Red blood cells were observed in thedamaged villi. The crypts were irregularly shaped and in had variousheights. The normal mouse showed smooth villi tips of the villi andnuclei of enterocytes were observed throughout the villus. The cryptswere similar in height and had a regular shape. The CP treated and TGF-αtreated mouse had normal appearing villi as described for the normalmouse. The crypts also appeared normal.

[0112] Further CP (10 μg/g) treated without TGF-α57 mice and CP (10μg/g) and 50 ng/g of TGF-α57 treated mice intestines when examined underhigher maginification showed severely injured crypt surfaces in the CPtreated mice due to cell death and necrosis. Red cells were visible atthe damaged surface indicating intestinal bleeding. In addition, theCP-treated mouse showed a loss of brush borders and very little of aglycocalyx or fuzzy coat. Globlet cells appeared interspersed, necroticand fewer in number than normal. The effect of TGF-α treatment showedprotection of the villa surface. Specifically, the epithelial cellsappeared normal with extended brush borders. The nuclei were verydensely stained and elongated.

[0113] The histological data is summarized in FIG. 4 that measuredaverage crypt height of the three groups of mice. TGFα57 and TGF-α (50a.a.) treatment (50 ng/g) was able to more-than-restore crypt heightloss from CP treatment.

[0114] An alcian blue staining method permited differentiation ofabsorptive cells and goblet cells. Goblet cell mucus is stained a bluecolor while the absorptive cells remain less stained. Stains ofintestine from normal mice, CP only treated (10 μg/g) and both CP (10μg/g) and TGFα57 (50 ng/g) treated mice showed significant differences.In the normal intestine each villus extended from the luminal surface tothe basal muscularis mucosal surface. Goblet cells were scattered andpredominated in the base of the villus whereas columnar absorptive cellslined the luminal surface. In the CP treated mouse, the alcian bluestaining showed villi that contained fewer number of goblet cells (thannormal). The injured absorptive and goblet cells were degenerating atthe tip of the villi and abundant secretory mucus material was stainedin the luminal surface. In the CP/TGF-α mouse, there were an increasednumber of goblet cells scattered throughout the villi. The intestinalvilli appeared normal with elongation. The majority of enterocytes didnot appear to be alcian blue stained positive. The luminal plasmamembranes of the villi were well protected by TGF-α treatment. Thenumber of goblet cells was counted on the average unit length ofintestine. TGFα treatment not only increased the number of goblet cellsbut also increased the number from CP treatment to a higher level thannormal intestine.

[0115] Accordingly, these data show the effects of TGFα , and thefunctional peptides having TGF-α activity from formula I, formula II,formula III, and formula IV having therapeutic activity to treat orprevent mucositis associated with cytotoxic drug therapy and forinflammatory bowel diseases. Moreover, the histological effect showingthat there was a prevention of mast cell degranulation, providesadditional data supporting the gastrointestinal applications for TGFα,and the functional peptides having TGF-α activity of formula I, formulaII, formula III, and formula IV.

EXAMPLE 4 Immune Related Diseases

[0116] In addition, TGF-α activity resulted in stimulation ofproliferation of select immune cells (particularly of the T celllineage) after administration to mice after immune-suppression of CPadministration. The stimulated immune cells were phenotypicallyidentified as CD4 positive T cells and double null CD4 negative CD8negative T cell progenitors. Thus, TGF-α activity (e.g., from TGFα57administration) resulted in generation of T-cells with characterisiticsthat regulated immune functions. Therefore, these data predict that TGFαactivity and the functional peptides of formula I, formula II, formulaIII, and formula IV will be effective in treating autoimmune diseases bymitigating over-inflammatory reactions. The in vivo activity of TGFα(and the functional peptides of formula I, formula II, formula III, andformula IV) to stimulate early T cell progenitors in the release of TH-1and TH-2 cytokines and this regulation of immune phenomena. Thestimulation of select immune cells, in particular cells of a T celllineage, was seen consistently in the mice who received CP and TGF-α57in lymphoid tissue, Peyers Patches and the spleen. Further, recruitmentof help via CD4 cells in some cases boosts immune system function ingeneral.

[0117] TGF-α administration prevented mast cell degranulation andsubsequent histamine release. In addition TGF-α has effectes indownregulating TNF-α receptors in vivo and downreglating IL-6 and MIP invivo, including blockign neutrophil trafficking. This is a parallelactivity that is in addition to the gastrointestinal anti-inflammatoryactivity and prevention of mucositis of TGFα (and the functionalpeptides of formula I, formula II, formula III, and formula IV)described herein.

EXAMPLE 5

[0118] In order to determine the effects of TGF-α polypeptides onweight-loss four groups of rats were tested. The experiment was designedto compare two of the peptides of TGF-α (SEQ ID NO:1 and SEQ ID NO:3) onweight-loss in the presence of a chemotherapeutic drug, cisplatin.

[0119] All animals were dosed over a period of 5 days. Group 1 animalsreceived cisplatin at 10 μg/g, Group 2 animals received cisplatin at 10μg/g plus TGF-α (SEQ ID NO:1) at 50 ng/g; Group 3 animals receivedcisplatin at 10 μg/g plus TGF-α57 (SEQ ID NO:3) at 50 ng/g; and Group 4animals received TGF-α (SEQ ID NO:1) at 50 ng/g. Following completion ofthe dosing protocols animals from each group were measure andorgans/tissues were harvested and placed in buffered formalin. Thetissues measured included lungs, spleens, kidneys, pancreas, intestinesand tongues.

[0120] In the 9 animals in group 1 (cisplatin treatment), the averageweight loss was 18.3%; in the 13 animals in groups 2 and 3(cisplatin+TGF) the average weight loss was 12.1%; and in the 6 animalsin group 4 (TGF alone) the average weight loss was 0.9% (FIG. 5).

[0121] In addition, studies of TGF-α for the treatment of diarrhea innon-human primates was also performed. A 6-year old non-human primateexhibiting chronic inflammatory-like gastrointestinal symptoms wastreated with TGF-α at 300 ng/g intraperitoneally once and subsequently50 ng/g S.C. for 6 days. The primate showed a steady increase in stoolconsistency and the monkey showed steady weight gain through thetreatment period (see Table 1 and 2). This weight gain was maintained atleast for several weeks post treatment. In addition, the reduction ofSEGs (see column 6, Table 1) neutrophils correlates with reduction ininflammation associated with neutrophil influx and concomitantpro-oinflammatory cytokines. No adverse effects were noted in hematologyor serum chemistries, or in the primates attitude, behavior or appetite.TABLE 1 Dual Time wt. hemoglobin pcv % wbc Seg Bands Lymph Monos EosBasos Abn. Cells Platelets CD4⁺ CD8⁺ CD8⁺ CD4⁺ 1 5.80 12.6 40.3 16.6 76%0% 19% 5% 0% 0% 0% 543 28.4% 56.3% 6.7% 2 6.33 11.1 38.0 20.2 69% 0% 20%10%  1% 0% 0% 567 3 6.74  9.2 32.1 10.3 61% 0% 34% 4% 1% 0% 0% 456 36.7%53.1% 6.3% 4 8.21 10.3 34.2  8.4 48% 0% 42% 9% 1% 0% 0% 534 31.3% 54.0%9.1% 5 8.93 10.8 37.1 11.2 46% 0% 45% 8% 0% 1% 0% 425 33.4% 49.8% 8.1% 69.42 11.5 38.1 15.5 56% 1% 31% 11%  1% 0% 0% 434 35.1% 50.3% 7.7%

[0122] TABLE 2 Total Albumin Total Alk ALT AST Time Na⁺ K⁺ Cl⁻ Glu BUNCreatine Protein Albumin corrected Billirubin Ca²⁺ Phos (GPT) (GOT) GGT1 133 3.3 87 73 45 0.6 8.6 4.9 5.9 1.2 11.6 103 8 26 57 2 144 4.8 95 5119 0.5 7.4 3.7 4.4 3 146 4.6 107 68 16 0.5 7.2 2.9 3.5 0.3 9.8 116 17 4589 4 150 4.4 107 50 19 0.7 7.1 2.9 3.5 0.3 9.7 130 25 34 91 5 148 4.0108 25 18 0.6 6.6 2.5 3.0 0.2 9.1 107 19 37 64 6 145 3.8 108 21 22 0.57.0 2.5 3.0 0.3 9.3 113 15 34 58

[0123] While the invention has been described in detail with referenceto certain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

1 6 1 50 PRT Homo sapiens 1 Val Val Ser His Phe Asn Asp Cys Pro Asp SerHis Thr Gln Phe Cys 1 5 10 15 Phe His Gly Thr Cys Arg Phe Leu Val GlnGlu Asp Lys Pro Ala Cys 20 25 30 Val Cys His Ser Gly Tyr Val Gly Ala ArgCys Glu His Ala Asp Leu 35 40 45 Leu Ala 50 2 50 PRT Rattus norvegicus 2Val Val Ser His Phe Asn Lys Cys Pro Asp Ser His Thr Gln Tyr Cys 1 5 1015 Phe His Gly Thr Cys Arg Phe Leu Val Gln Glu Glu Lys Pro Ala Cys 20 2530 Val Cys His Ser Gly Tyr Val Gly Val Arg Cys Glu His Ala Asp Leu 35 4045 Asp Ala 50 3 57 PRT Artificial sequence A modified human TGF-alphasequence 3 Ser Leu Ser Leu Pro Ala Met Val Val Ser His Phe Asn Asp CysPro 1 5 10 15 Asp Ser His Thr Gln Phe Cys Phe His Gly Thr Cys Arg PheLeu Val 20 25 30 Gln Glu Asp Lys Pro Ala Cys Val Cys His Ser Gly Tyr ValGly Ala 35 40 45 Arg Cys Glu His Ala Asp Leu Leu Ala 50 55 4 11 PRTArtificial sequence Artificial peptide sequence 4 Xaa Cys His Ser XaaXaa Xaa Xaa Xaa Xaa Cys 1 5 10 5 7 PRT Artificial sequence Artificialpeptide sequence 5 Xaa His Xaa Xaa Xaa Xaa Xaa 1 5 6 18 PRT Artificialsequence Artificial peptide sequence 6 Xaa Cys His Ser Xaa Xaa Xaa XaaXaa Xaa Cys Xaa His Xaa Xaa Xaa 1 5 10 15 Xaa Xaa

What is claimed:
 1. A polypeptide comprising a peptide having a sequenceNH₂-X_(1a)-Cys-His-Ser-X_(1b)-X₂-X_(1a)-X_(1b)-X_(1a)-X₃-Cys-COOH (SEQID NO:4) wherein X_(1a) and X_(1b) are independently Val, Gly or Ala; X₂is Tyr or Phe; X₃ is Arg or Lys; and the two Cys moieties are linked viaa disulfide bond to form an at least 11-amino acid functional peptidehaving TGF-α activity.
 2. The polypeptide of claim 1, wherein at leastone or more of the following amino acids are linked to the C-terminalCys moiety of SEQ ID NO:4: -X₄-His-X_(1c)-X₄-X₅-X₆-X_(1c) (SEQ ID NO:5)wherein X₄ is Glu or Asp; X₅ is Leu or Ile; and X₆ is Asp or Glu.
 3. Thepolypeptide of claim 2, wherein X_(1a) is Val, X_(1b) is Gly and X_(1c)is Ala.
 4. The polypeptide of claim 2, wherein X₂ is Tyr and X₃ is Arg.5. The polypeptide of claim 2, wherein the functional peptide is 18amino acids in length and X_(1a) is Val, X_(1b) is Gly, X_(1c) is Alaand X₄ is Gly.
 6. A method of treating a subject having or at risk ofexhibiting weight-loss comprising administering to the subject atransfonning growth factor-alpha (TGF-α) polypeptide in an effectiveamount to inhibit or reduce weight-loss in the subject.
 7. The method ofclaim 6, wherein the polypeptide has an amino acid sequence as set forthin SEQ ID NO:1.
 8. The method of claim 6, wherein the polypeptide has anamino acid sequence as set forth in SEQ ID NO:3.
 9. The method of claim6, wherein the polypeptide comprises a peptide having a sequenceNH₂-X_(1a)-Cys-His-Ser-X_(1b)-X₂-X_(1a)-X_(1b)-X_(1a)-X₃-Cys COOH (SEQID NO:4) wherein X_(1a) and X_(1b) are independently Val, Gly or Ala; X₂is Tyr or Phe; X₃ is Arg or Lys; and the two Cys moieties are linked viaa disulfide bond to form an at least 11-amino acid functional peptidehaving TGF-α activity.
 10. The method of claim 9, wherein at least oneor more of the following amino acids are linked to the C-terminal Cysmoiety of SEQ ID NO:4:-X₄-His-X_(1c)-X₄-X₅-X6-X_(1c) (SEQ ID NO:5)wherein X₄ is Glu or Asp; X₅ is Leu or Ile; and X₆ is Asp or Glu. 11.The method of claim 10, wherein X_(1a) is Val, X_(1b) is Gly and X_(1c)is Ala.
 12. The method of claim 10, wherein X₂ is Tyr and X₃ is Arg. 13.The method of claim 10, wherein the functional peptide is 18 amino acidsin length and X_(1a) is Val, X_(1b) is Gly, X_(1c) is Ala and X₄ is Gly.14. The method of claim 6, wherein the weight-loss is the result of adisease or disorder.
 15. The method of claim 6, wherein the weight-lossis associated with treatment with of the subject with a chemotherapeuticagent.
 16. The method of claim 15, wherein the chemotherapeutic agent isselected from the group consisting of carmustine (BCNU), chlorambucil(Leukeran), cisplatin (Platinol), Cytarabine, doxorubicin (Adriamycin),fluorouracil (5-FU), methoxetrate (Mexate), taxol, CPT111, etoposide,and plicamycin (Mithracin).
 17. The method of claim 15, wherein thedisease or disorder is ARC or AIDS.
 18. The method of claim 6, whereinthe subject is a mammal.
 19. The method of claim 18, wherein the mammalis a human.
 20. A method of increasing the body weight of a subjectcomprising administering to the subject prior to, simultaneously with,or substantially following weight-loss a transforming growthfactor-alpha (TGF-α) polypeptide in an effective amount to increase ormaintain the weight of the subject.
 21. The method of claim 20, whereinthe polypeptide has an amino acid sequence as set forth in SEQ ID NO:1.22. The method of claim 20, wherein the polypeptide has an amino acidsequence as set forth in SEQ ID NO:3.
 23. The method of claim 20,wherein the polypeptide comprises a peptide having a sequenceNH₂-X_(1a)-Cys-His-Ser-X_(1b)-X₂-X_(1a)-X_(1b)-X_(1a)-X₃-Cys COOH (SEQID NO:4) wherein X_(1a) and X_(1b) are independently Val, Gly or Ala; X₂is Tyr or Phe; X₃ is Arg or Lys; and the two Cys moieties are linked viaa disulfide bond to form an at least 11-amino acid functional peptidehaving TGF-α activity.
 24. The method of claim 23, wherein at least oneor more of the following amino acids are linked to the C-terminal Cysmoiety of SEQ ID NO:4:-X₄-His-X_(1c)-X₄-X₅-X₆-X_(1c) (SEQ ID NO:5)wherein X₄ is Glu or Asp; X₅ is Leu or Ile; and X6 is Asp or Glu. 25.The method of claim 24, wherein X_(1a) is Val, X_(1b) is Gly and X_(1c)is Ala.
 26. The method of claim 24, wherein X₂ is Tyr and X₃ is Arg. 27.The method of claim 24, wherein the functional peptide is 18 amino acidsin length and X_(1a) is Val, X_(1b) is Gly, X_(1c) is Ala and X₄ is Gly.28. The method of claim 20, wherein the weight-loss is the result of adisease or disorder.
 29. The method of claim 20, wherein the weight-lossis associated with treatment of the subject with a chemotherapeuticagent.
 30. The method of claim 29, wherein the chemotherapeutic agent isselected from the group consisting of carmustine (BCNU), chlorambucil(Leukeran), cisplatin (Platinol), Cytarabine, doxorubicin (Adriamycin),fluorouracil (5-FU), methoxetrate (Mexate), taxol, CPT111, etoposide,and plicamycin (Mithracin).
 31. The method of claim 28, wherein thedisease or disorder is ARC or AIDS.
 32. The method of claim 20, whereinthe subject is a mammal.
 33. The method of claim 32, wherein the mammalis a human.
 34. A pharmaceutical composition comprising a polypeptidehaving a sequenceNH₂-X_(1a)-Cys-His-Ser-X_(1b)-X₂-X_(1a)-X_(1b)-X_(1a)-X₃-Cys COOH (SEQID NO:4) wherein X_(1a) and X_(1b) are independently Val, Gly or Ala; X₂is Tyr or Phe; X₃ is Arg or Lys; and the two Cys moieties are linked viaa disulfide bond to form an at least 11-amino acid functional peptidehaving TGF-α activity, and a pharmaceutically acceptable carrier. 35.The pharmaceutical composition of claim 34, wherein at least one or moreof the following amino acids are linked to the C-terminal Cys moiety ofSEQ ID NO:4:-X₄-His-X_(1c)-X₄-X₅-X6-X_(1c) (SEQ ID NO:5) wherein X₄ isGlu or Asp; X₅ is Leu or Ile; and X₆ is Asp or Glu.
 36. Thepharmaceutical composition of claim 34, wherein X_(1a) is Val, X_(1b) isGly and X_(1c) is Ala.
 37. The pharmaceutical composition of claim 35,wherein X₂ is Tyr and X₃ is Arg.
 38. The pharmaceutical composition ofclaim 35, wherein the functional peptide is 18 amino acids in length andX_(1a) is Val, X_(1b) is Gly, X_(1c) is Ala and X₄ is Gly.
 39. A methodfor treating a neurodegenerative disease in a subject comprisingadministering to the subject a transforming growth factor-alpha (TGF-α)polypeptide in an effective amount to modulate the neurodegenerativedisease.
 40. The method of claim 39, wherein the polypeptide has anamino acid sequence as set forth in SEQ ID NO:1.
 41. The method of claim39, wherein the polypeptide has an amino acid sequence as set forth inSEQ ID NO:3.
 42. The method of claim 39, wherein the polypeptide has asequence NH₂-X_(1a)-Cys-His-Ser-X_(1b)-X₂-X_(1a)-X_(1b)-X_(1a)-X₃-CysCOOH (SEQ ID NO:4) wherein X_(1a) and X_(1b) are independently Val, Glyor Ala; X₂ is Tyr or Phe; X₃ is Arg or Lys; and the two Cys moieties arelinked via a disulfide bond to form an at least 11-amino acid functionalpeptide having TGF-α activity.
 43. The method of claim 42, wherein atleast one or more of the following amino acids are linked to theC-terminal Cys moiety of SEQ ID NO:4:-X₄-His-X_(1c)-X₄-X₅-X₆-X_(1c) (SEQID NO:5) wherein X₄ is Glu or Asp; X₅ is Leu or Ile; and X₆ is Asp orGlu.
 44. The method of claim 43, wherein X_(1a) is Val, X_(1b) is Glyand X_(1c) is Ala.
 45. The method of claim 43, wherein X₂ is Tyr and X₃is Arg.
 46. The method of claim 43, wherein the functional peptide is 18amino acids in length wherein X_(1a) is Val, X_(1b) is Gly, X_(1c) isAla and X₄ is Gly.
 47. A method for treating a CNS disease or disordercomprising administering to a subject a polypeptide having a sequence asset forth in SEQ ID NO:3, or a polypeptide having a sequenceNH₂-X_(1a)-Cys-His-Ser-X_(1b)-X₂-X_(1a)-X_(1b) -X_(1a)-X₃-Cys COOH (SEQID NO:4) wherein X_(1a) and X_(1b) are independently Val, Gly or Ala; X₂is Tyr or Phe; X₃ is Arg or Lys; and the two Cys moieties are linked viaa disulfide bond to form an at least 11-amino acid functional peptidehaving TGF-α activity.
 48. The method of claim 47, wherein the CNSdisease or disorder is selected from the group consisting of CNSischemia, spinal cord injury, MS, and retinal injury.
 49. The method ofclaim 47, wherein at least one or more of the following amino acids arelinked to the C-terminal Cys moiety of SEQ IDNO:4:-X₄-His-X_(1c)-X₄-X₅-X₆-X_(1c) (SEQ ID NO:5) wherein X₄ is Glu orAsp; X₅ is Leu or Ile; and X₆ is Asp or Glu.
 50. The method of claim 49,wherein X_(1a) is Val, X_(1b) is Gly and X_(1c) is Ala.
 51. The methodof claim 49, wherein X₂ is Tyr and X₃ is Arg.
 52. The method of claim49, wherein the functional peptide is 18 amino acids in length andwherein X_(1a) is Val, X_(1b) is Gly, X_(1c) is Ala and X₄ is Gly.
 53. Amethod for enhancing hematopoiesis during cytotoxic orimmune-suppressing therapy, comprising administering a TGFα polypeptidein an effective amount to enhance hematopoeisis.
 54. The method of claim53, wherein the polypeptide has an amino acid sequence as set forth inSEQ ID NO:1.
 55. The method of claim 53, wherein the polypeptide has anamino acid sequence as set forth in SEQ ID NO:3.
 56. The method of claim53, wherein the polypeptide has a sequenceNH₂-X_(1a)-Cys-His-Ser-X_(1b)-X₂-X_(1a)-X_(1b)-X_(1a)-X₃-Cys-COOH (SEQID NO:4) wherein X_(1a) and X_(1b) are independently Val, Gly or Ala; X₂is Tyr or Phe; X₃ is Arg or Lys; and the two Cys moieties are linked viaa disulfide bond to form an at least 11-amino acid functional peptidehaving TGF-α activity.
 57. The method of claim 56, wherein at least oneor more of the following amino acids are linked to the C-terminal Cysmoiety of SEQ ID NO:4:-X₄-His-X_(1c)-X₄-X₅-X₆-X_(1c) (SEQ ID NO:5)wherein X₄ is Glu or Asp; X₅ is Leu or Ile; and X₆ is Asp or Glu. 58.The method of claim 57, wherein X_(1a) is Val, X_(1b) is Gly and X_(1c)is Ala.
 59. The method of claim 57, wherein X₂ is Tyr and X₃ is Arg. 60.The method of claim 57, wherein the functional peptide is 18 amino acidsin length wherein X_(1a) is Val, X_(1b) is Gly, X_(1c) is Ala and X₄ isGly.
 61. The method of claim 53, further comprising administering asecond hematopoietic growth factor agent to stimulate the generation ofmore mature hematopoietic precursor cells, wherein the secondhematopoietic growth factor is selected from the group consisting oferythropoietin, thrombopoietin, G-CSF (granulocyte colony stimulatingfactor), and GM-CSF (granulocyte macrophage colony stimulating factor).62. A method for treating or preventing mucositis of thegastrointestinal tract in a subject comprising administering a TGFαpolypeptide in an amount effective to inhibit or prevent oral orintestinal mucositis in the subject.
 63. The method of claim 62, whereinthe polypeptide has an amino acid sequence as set forth in SEQ ID NO:1.64. The method of claim 62, wherein the polypeptide has an amino acidsequence as set forth in SEQ ID NO:3.
 65. The method of claim 62,wherein the polypeptide has a sequenceNH₂-X_(1a)-Cys-His-Ser-X_(1b)-X₂-X_(1a)-X_(1b)-X_(1a)-X₃-Cys COOH (SEQID NO:4) wherein X_(1a) and X_(1b) are independently Val, Gly or Ala; X₂is Tyr or Phe; X₃ is Arg or Lys; and the two Cys moieties are linked viaa disulfide bond to form an at least 11-amino acid functional peptidehaving TGF-α activity.
 66. The method of claim 65, wherein at least oneor more of the following amino acids are linked to the C-terminal Cysmoiety of SEQ ID NO:4:-X₄-His-X_(1c)-X₄-X₅-X₆-X_(1c) (SEQ ID NO:5)wherein X₄ is Glu or Asp; X₅ is Leu or Ile; and X₆ is Asp or Glu. 67.The method of claim 66, wherein X_(1a) is Val, X_(1b) is Gly and X_(1c)is Ala.
 68. The method of claim 66, wherein X₂ is Tyr and X₃ is Arg. 69.The method of claim 66, wherein the functional peptide is 18 amino acidsin length wherein X_(1a) is Val, X_(1b) is Gly, X_(1c) is Ala and X₄ isGly.
 70. The method of claim 62, wherein the oral or intestinalmucositis results from cytotoxic or immune-suppressing therapy.
 71. Themethod of claim 62, wherein the mucositis results from ARC or AIDS. 72.The method of claim 62, wherein the subject is a mammal.
 73. The methodof claim 72, wherein the mammal is a human.
 74. A compound as shown informula III: loop peptide N-terminus-linker-cyclic C₄H₈N₂-linker-looppeptide N-terminus   (III) wherein the linker moiety is designed to linkthe N-terminus of the loop peptide to a nitrogen atom of the ring C₄H₈N₂and wherein the loop peptide has a sequenceNH₂-X_(1a)-Cys-His-Ser-X_(1b)-X₂-X_(1a)-X_(1b)-X_(1a)-X₃-Cys COOH (SEQID NO:4) wherein X_(1a) and X_(1b) are independently Val, Gly or Ala; X₂is Tyr or Phe; X₃ is Arg or Lys; and the two Cys moieties are linked viaa disulfide bond to form an at least 11-amino acid functional peptidehaving TGF-α activity, wherein said compound functions as a TGF-αmimetic.
 75. The compound of claim 74, wherein at least one or more ofthe following amino acids are linked to the C-terminal Cys moiety of SEQID NO:4: -X₄-His-X_(1c)-X₄-X₅-X₆-X_(1c) (SEQ ID NO:5) wherein X₄ is Gluor Asp; X₅ is Leu or Ile; and X₆ is Asp or Glu.
 76. The compound ofclaim 75, wherein X_(1a) is Val, X_(1b) is Gly and X_(1c) is Ala. 77.The compound of claim 75, wherein X₂ is Tyr and X₃ is Arg.
 78. Thecompound of claim 75, wherein the functional peptide is 18 amino acidsin length wherein X_(1a) is Val, X_(1b) is Gly, X_(1c) is Ala and X₄ isGly.
 79. The compound of claim 74, wherein the linker group isindependently selected from the group consisting of substituted orunsubstituted C₁₋₆ alkyl, substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₁₋₆ alkoxy, xylenyl, wherein thesubstitutions are selected from the group consisting of oxo, epoxyl,hydroxyl, chloryl, bromyl, fluoryl, and amino.
 80. A method for treatinginflammatory bowel disease, colitis, and Chron's Disease of thegastrointestinal tract, comprising administering a TGFα polypeptide inan effective amount to inhibit or ameliorate the disease.
 81. The methodof claim 80, wherein the polypeptide has an amino acid sequence as setforth in SEQ ID NO:1.
 82. The method of claim 80, wherein thepolypeptide has an amino acid sequence as set forth in SEQ ID NO:3. 83.The method of claim 80, wherein the polypeptide has a sequenceNH₂-X_(1a)-Cys-His-Ser-X_(1b)- X₂-X_(1a)-X_(1b)-X_(1a)- X₃-Cys COOH (SEQID NO:4) wherein X_(1a) and X_(1b) are independently Val, Gly or Ala; X₂is Tyr or Phe; X₃ is Arg or Lys; and the two Cys moieties are linked viaa disulfide bond to form an at least 11-amino acid functional peptidehaving TGF-α activity.
 84. The method of claim 83, wherein at least oneor more of the following amino acids are linked to the C-terminal Cysmoiety of SEQ ID NO:4:-X₄-His-X_(1c)-X₄-X₅-X₆-X_(1c) (SEQ ID NO:5)wherein X₄ is Glu or Asp; X₅ is Leu or Ile; and X₆ is Asp or Glu. 85.The method of claim 84, wherein X_(1a) is Val, X_(1b) is Gly and X_(1c)is Ala.
 86. The method of claim 84, wherein X₂ is Tyr and X₃ is Arg. 87.The method of claim 84, wherein the functional peptide is 18 amino acidsin length and wherein X_(1a) is Val, X_(1b) is Gly, X_(1c) is Ala, andX₄ is Gly.
 88. A method for treating or preventing an inflammatoryreaction of an autoimmune disease in a subject comprising administeringa TGFα polypeptide to the subject in an effective amount to inhibit orameliorate the inflammatory reaction.
 89. The method of claim 88,wherein the polypeptide has an amino acid sequence as set forth in SEQID NO:1.
 90. The method of claim 88, wherein the polypeptide has anamino acid sequence as set forth in SEQ ID NO:3.
 91. The method of claim88, wherein the polypeptide has a sequenceNH₂-X_(1a)-Cys-His-Ser-X_(1b)-X₂-X_(1a)-X_(1b)-X_(1a)-X₃-Cys COOH (SEQID NO:4) wherein X_(1a) and X_(1b) are independently Val, Gly or Ala; X₂is Tyr or Phe; X₃ is Arg or Lys; and the two Cys moieties are linked viaa disulfide bond to form an at least 11-amino acid functional peptidehaving TGF-α activity.
 92. The method of claim 91, wherein at least oneor more of the following amino acids are linked to the C-terninal Cysmoiety of SEQ ID NO:4:-X₄-His-X_(1c)-X₄-X₅-X₆-X_(1c) (SEQ ID NO:5)wherein X₄ is Glu or Asp; X₅ is Leu or Ile; and X6 is Asp or Glu. 93.The method The method of claim 92, wherein X_(1a) is Val, X_(1b) is Glyand X_(1c) is Ala.
 94. The method of claim 92, wherein X₂ is Tyr and X₃is Arg.
 95. The method of claim 92, wherein the functional peptide is 18amino acids in length wherein X_(1a) is Val, X_(1b) is Gly, X_(1c) isAla and X₄ is Gly.
 96. The method of claim 88, wherein the autoimmunediseases are selected from the group consisting of Type II (Juvenile)Diabetes, rheumatoid arthritis, lupus, glomerular nephritis, andmultiple sclerosis.
 97. The method of claim 88, wherein the subject is amammal.
 98. The method of claim 97, wherein the mammal is a human.